Page 1 October 2014 ZEN 2 (black edition)
Page 2 Violations will entail an obligation to pay compensation. All rights reserved in the event of granting of patents or registration of a utility model. Issued by Carl Zeiss Microscopy GmbH Carl-Zeiss-Promenade 10 07745 Jena, Germany microscopy@zeiss.com www.zeiss.com/microscopy...
Page 3 INTRODUCTION LSM 880 ZEISS How to make best use of the LSM 880 and LSM 880 NLO operating instructions: A few symbols in these operating instructions will help you to recognize the nature and purpose of information immediately: The WARNING symbol warns against hazards for the user that might arise when operating the laser.
Page 4 INTRODUCTION ZEISS LSM 880 Contents System Operation In this section you will find the most important steps and procedures of the LSM menu structure. The step-by-step description how to get an image will be shown by typical application examples including the WINDOWS graphic user environment.
Page 5 GDD can still be moving. This can be set via database. Contact your ZEISS representative on details if that function is of interest for you.
Page 6 IMPORTANT NOTES ZEISS LSM 880 • The functions Crop, Split, Delete and Copy used in the file browser menu requires some time for GUI update. • When using the channel unmixing function for Airyscan data, the histogram of the resulting image still refers to the Airyscan data set.
Page 7: Table Of Contents
Principle of Laser Scanning Microscopy ..............10 Principle of Fluorescence Correlation Spectroscopy ........... 11 Principle of Airyscanning ..................... 12 Optical Diagram of the LSM 880 (Schematic) ............. 13 Performance Features of the LSM 880 ................ 14 Microscope Equipment of the LSM 880 ..............15 Computer Hardware and Software ................
Page 8 CHAPTER 1 - SYSTEM OPERATION ZEISS Contents LSM 880 4.5.3 Help – About ........................37 Main Toolbar ........................ 38 Status Bar ........................40 Left Tool Area ....................... 41 4.8.1 Tool Groups and Tools ..................... 43 4.8.2 Context Menu of the Left Tool Area ................. 44 Center Screen Area .......................
Page 9 CHAPTER 1 - SYSTEM OPERATION LSM 880 Contents ZEISS 5.2.9.1 Defining Channels and Tracks ..................116 5.2.9.2 Ratio Channels ......................120 5.2.9.3 Using GaAsP Detectors and the temperature controlled side PMT for Photon Counting Imaging ........................121 5.2.9.4 Channel Settings for Airyscan ..................124 5.2.10...
Page 10 CHAPTER 1 - SYSTEM OPERATION ZEISS Contents LSM 880 5.3.4 Setup – Fit ........................192 5.3.4.1 Defining a New Model ....................194 5.3.4.2 Correlation Options ....................... 195 5.3.4.3 Model Equations ......................196 5.3.4.4 Definition of a Model ....................213 5.3.4.5 PCH ..........................
Page 11 CHAPTER 1 - SYSTEM OPERATION LSM 880 Contents ZEISS 5.4.12 Processing – HDR-imaging .................... 321 5.4.13 Processing – Stitch ......................325 5.4.14 Processing – PALM ......................326 5.4.14.1 PALM – Convert to Image ..................... 327 5.4.14.2 PALM – Import PALM Molecules ................... 328 5.4.14.3 PALM –...
Page 12 CHAPTER 1 - SYSTEM OPERATION ZEISS Contents LSM 880 5.5.5.3 System Options – Hardware ................... 411 5.5.5.4 System Options – Image Display ..................412 5.5.6 Options – FCS Options ....................413 5.5.6.1 FCS Options – Data ....................... 413 5.5.6.2 FCS Options – Measure ....................417 5.5.6.3...
Page 13 CHAPTER 1 - SYSTEM OPERATION LSM 880 Contents ZEISS 6.11 Colocalization View ....................472 6.11.1 How a Scatter Diagram is Generated ................474 6.11.2 Quantitative Colocalization Parameters Shown in the Data Table ........475 6.11.3 Colocalization Coefficients .................... 475 6.11.4 Overlap Coefficient, Overlap Coefficient after Manders ..........
Page 14: Purpose
The installation of the software for the Laser Scanning Microscope and the basic settings of the equipment components are carried out by ZEISS service staff. This job includes the creation of a customized software configuration in line with the specific hardware components of the customer's microscope system.
Page 15: Backup
The following user-generated files should be included in a backup procedure controlled and carried out on a regular basis by the user (keep directory structure): − Carl Zeiss Image files: *.czi − LSM Image files: *.lsm − Exported images: *.* (*.Tiff, *.LSM-Tiff, *.BMP, …) −...
Page 16: Hardware Aspects
CHAPTER 1 - SYSTEM OPERATION ZEISS Hardware Aspects LSM 880 Hardware Aspects Principle of Laser Scanning Microscopy To yield information on their inner structure by conventional transmitted-light microscopy, specimens have to be very thin and translucent; otherwise image definition will be poor. In many cases it is a problem to satisfy these requirements.
Page 17: Principle Of Fluorescence Correlation Spectroscopy
CHAPTER 1 - SYSTEM OPERATION LSM 880 Hardware Aspects ZEISS Principle of Fluorescence Correlation Spectroscopy Fluorescence Correlation Spectroscopy (FCS) analyzes the diffusion time of molecules and their differences if they have bound together. This is done by fluctuation analysis of fluorescence-labeled molecules within a well defined volume element.
Page 18: Principle Of Airyscanning
CHAPTER 1 - SYSTEM OPERATION ZEISS Hardware Aspects LSM 880 Principle of Airyscanning The Airyscan detector consists of 32 elements based on GaAsP PMT technology with each element detecting the emission signal of a focal spot much smaller than the typical 1 Airy volume.
Page 19: Optical Diagram Of The Lsm 880 (Schematic)
Fig. 4 Optical path on an inverted microscope, schematic (2 PMT / 32 array configuration) The diagram above is a schematic representation of the LSM 880 system with attached Airyscan and BiG.2 module. Laser light is focused onto the specimen through an objective in a diffraction-limited mode. Light emitted at the focal plane and at planes below and above it is directed via an XY scanner onto a main dichroic beam splitter (MDBS), which separates the emissions from the excitation light.
Page 20: Performance Features Of The Lsm 880
The highly integrated system design makes for the shortest possible optical paths, top-grade optical precision and high stability. The Airyscan module can be mounted to the LSM 880 scan head of any configuration and with upright or inververted microscopes. The BiG.2 module is either directly attached to the scan head and can be used in combination with upright and inverse microscope stands.
Page 21: Microscope Equipment Of The Lsm 880
ZEISS Microscope Equipment of the LSM 880 LSM 880 is attached to either the rear port or side port of the inverted microscope Axio Observer.Z1 or to the upright microscopes Axio Imager.Z2 or Axio Examiner.Z1. Only the differences from the delivered operating manual "Axio Observer.Z1" will be explained here.
Page 22 Photo equipment The stand does not feature an integrated SLR-port, but microscope cameras as described in the Axio Observer.Z1 and LSM 880 NLO operation manuals can be used. Camera adaptation The camera port at the side or the front can be used as described in the Axio Observer.Z1 operation manual.
Page 23: Computer Hardware And Software
ZEISS Computer Hardware and Software The LSM 880 (NLO) Airyscan / BiG.2 are controlled via a standard high-end Pentium / Xeon PC. Linking to the electronic control system is made via Gigabit Ethernet interface. The PC comes with the WINDOWS operating system.
Page 24: Startup And Shutdown Of The System
CHAPTER 1 - SYSTEM OPERATION ZEISS Startup and Shutdown of the System LSM 880 Startup and Shutdown of the System Startup of the System The LSM system is equipped with a main power switch and two further switches labeled System/PC and Components that are located at the front of the System Electronic Rack.
Page 25: Additional Software Tools
System Maintenance Tool icon With a calibration objective and correct system configuration, the maintenance tool allows convenient self adjustment of the LSM 880 systems. The optical beam path, relative pinhole position and scanner adjustment can be set and checked automatically on the systems.
Page 26: Starting Zen
(Fig. 6). It displays the ZEN symbol in the upper part, the designation of the current ZEN software version (lower left) and the Zeiss icon (lower right). − Once the program is loaded the Login ZEN dialog Fig. 6 Starting the ZEN software –...
Page 27 CHAPTER 1 - SYSTEM OPERATION LSM 880 Startup and Shutdown of the System ZEISS The Login ZEN dialog allows you to boot the Software in different modes. If the Boot Status is closed, the options are Start System and Image Processing.
Page 28 ZEISS Startup and Shutdown of the System LSM 880 − If Recent… is pressed the last couple of databases that were in use will be displayed (Fig. 12). Click on the one you want to select. By pressing the Choose… button Windows Explorer will open. Search for the folder file the database is stored and and open it.
Page 29: Shutdown Procedure
CHAPTER 1 - SYSTEM OPERATION LSM 880 Startup and Shutdown of the System ZEISS Shutdown Procedure Never shut down the computer by its main switch while the ZEN software is still active, or else you will lose the currently set operating parameters and the images just scanned.
Page 30 CHAPTER 1 - SYSTEM OPERATION ZEISS Startup and Shutdown of the System LSM 880 The Software can be shut down from File menu of the Main toolbar by selecting Exit (Fig. 13) or by pressing the Window Close button (symbolized by a cross) of the ZEN window.
Page 31: Switching System Power Off
Power Supply switch. 5 minutes after computer shutdown set the power remote switches System/PC and Components or the Main switch on the System Electronic Rack to position OFF. This puts your LSM 880 including the computer off power. 10/2014 V_01 000000-2071-464...
Page 32: Introduction To The Software Application Layout
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Introduction to the Software Application Layout Overview on the Screen Layout Fig. 15 ZEN Main Application window after Startup with empty image container ig. 16 ZEN Main Application window after Startup with several images loaded...
Page 33: Introduction To Zen
Introduction to the Software Application Layout ZEISS Introduction to ZEN ZEN - Efficient Navigation - is the new software for the LSM Family from ZEISS. With the launch of this software in 2007 ZEISS sets new standards in application-friendly software for Laser Scanning Microscopy.
Page 34 CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Fig. 17 Show all mode The Show all concept ensures that tool panels are never more complex than needed. In the basic mode of the tools Show all is deactivated and the tools show only the most relevant functions, covering approximately 80 % of the application.
Page 35 CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS Fig. 18 ZEN window Layout configuration More features of ZEN include: − The user can add more columns to the Left Tool Area or detach individual tools to position them anywhere on the monitor.
Page 36: Function Elements
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Function Elements Function element Description / Explanation Tool group Tool (closed) or tool window (opened) Panel (e.g.: Speed panel) − Field with a subset of tools of a tool window List box or selection box −...
Page 37 CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS Slider with input ("spin") box and arrows − Setting of numbers in the relevant input box by moving the slider or clicking on the arrow buttons or clicking on the slider and moving via the arrow keys of the keyboard.
Page 38: Application Bar
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Application Bar The application bar includes the following control elements of the ZEN software application window: Starts the online help Minimizes the application window Switches between maximized-window mode and compressed-window mode...
Page 39 CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS Menu overview File View Acquisition Maintain Macro Tools Window Paste Image New Acquisition Text View New Acquisition Set Spline Macro Close Pastes an image Document Displays the...
Page 40: File
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 For detailed information about the image acquisition functions see section Acquisition Tab. The functionalities of the Macro menu are described in CHAPTER 2: MACROS AND VISUAL BASIC.
Page 41: Maintain
CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS 4.5.2 Maintain The Maintain menu from the Menu bar contains additional modules to check and guarantee the interference-free operation of all the software and hardware components of the LSM 710 and LSM 780 systems.
Page 42: Camera
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 4.5.2.2 Camera This function allows the user to adjust the white balance and color balance of a connected camera. • Click on Camera in the Maintain list.
Page 43: Help - About
CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS 4.5.3 Help – About The About window can be accessed via the Help item in the menu bar clicking About opens the following dialog: Fig. 25...
Page 44: Main Toolbar
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Main Toolbar Fig. 26 Main toolbar, left side Fig. 27 Main toolbar, right side Workspace Configuration This function allows loading, saving or deleting a workspace configuration. These workspace...
Page 45 CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS • To load a workspace configuration, click on the button. The following list box will appear: Selecting on of the list entries will load the respective configuration.
Page 46: Status Bar
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Status Bar Fig. 28 Status bar Progress bar Shows an overview of all running processes. If only one process is running the details of this one are shown.
Page 47: Left Tool Area
CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS Left Tool Area Main Tool tabs Switches between the Locate, Acquisition, Processing and Maintain main tools to operate the included tool Action buttons Action buttons are only available for the Acquisition Main tool to control the image acquisition process.
Page 48 CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Multidimensional Acquisition: Tools for all multidimensional imaging The tools of this tool group will only appear if selected in the selection panel below the start buttons.
Page 49: Tool Groups And Tools
CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS 4.8.1 Tool Groups and Tools Multiple Columns Layout of tool groups This function allows moving one or several tool groups in a second or third column of the Left Tool Area.
Page 50: Context Menu Of The Left Tool Area
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 4.8.2 Context Menu of the Left Tool Area There are 2 different context menus in the Left Tool Area with different functionalities available. New Acquisition Document opens an empty acquisition document in the active image container.
Page 51: Center Screen Area
CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS Center Screen Area The Center Screen Area is used for displaying scanned images or to show images in the available image views. Using the context menu of the Center Screen Area by clicking the background of a container, the view of this area can be varied individually.
Page 52: Context Menu Of The Center Screen Area
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 Image tabs For each opened image one image tab is shown in the header Fig. 35 Image tabs of the actual container. Three modes can be selected using the context menu of the Center Screen Area (see also section Context Menu of the Center Screen Area).
Page 53 CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS Arrow down button Clicking on this button shows a list with all opened images for fast image selection. Using the drag and drop function, one or several images can be moved into another opened container.
Page 54: Right Tool Area
CHAPTER 1 - SYSTEM OPERATION ZEISS Introduction to the Software Application Layout LSM 880 4.10 Right Tool Area The Right Tool Area is used for displaying and handling the opened images, e.g. save or close. The view of this area can be varied individually, e.g.
Page 55 CHAPTER 1 - SYSTEM OPERATION LSM 880 Introduction to the Software Application Layout ZEISS Big View button Shows images with the textual characters and a big image preview. Save Status icon This icon appears in the image that is not saved yet.
Page 56: Left Tool Area And Hardware Control Tools
Visual and camera inspection in transmitted and epifluorescence light Acquisition Imaging with LSM or ELYRA Fig. 40 Locate tab Fluorescence Correlation Spectroscopy (LSM 880 only). Processing Processing superresolution and LSM images Maintain Setting Imaging and Firmware parameters By pressing the arrow left symbol of the Expand slider on the right border, the left tool area will collapse to the left side.
Page 57: Locate Tab
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Locate Tab Locate contains all functions of the microscope for visual inspection of the sample through the eyepieces (Fig. 41). You can open the shutters for Transmitted Light Fig.
Page 58 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 To delete a configuration press the Delete button ). This opens the Delete configuration panel (Fig. 43). Select ocular configuration from Configurations list by highlighting it or search for it by typing its name in the Search field.
Page 59: Microscope Control Tool For Axio Observer.z1
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS These microscope functions can also be operated directly on the microscope stand via assigned buttons or through the touch screen, if available. In that case, any changes are recorded by the software and displayed in the relevant windows / panels.
Page 60 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 HAL transmitted light Open/Close shutter button: The shutter for transmitted light from the HAL lamp is switched on and off by repeated pressing (Fig. 49).
Page 61 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Focus button: If the focus button is pressed, the Focus control panel will appear (Fig. 52). The Z-Position display box shows the current z-position. The number can be edited by typing or using the arrowheads.
Page 62 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Tube Lens button: The tube lens can be selected via a graphical pop- up menu (Fig. 55). Lens 1x is used for ocular observation and widefield imaging using an arc lamp, Lens LSM is used for laser scanning imaging and Lens 1.6x is used for widefield imaging using...
Page 63 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Visual inspection of the cells via binoculars • In the main toolbar, activate the Locate tab. • Choose from the Locate tab the Microscope Control tool.
Page 64 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Conventional setting of the microscope Axio Observer.Z1 The Recording of microscope settings works as described for the microscope Axio Imager. conventional setting Axio Observer.Z1, proceed as follows: •...
Page 65 Left Tool Area and Hardware Control Tools ZEISS Fig. 59 LSM 880 with Axio Observer.Z1 Microscope settings on Axio Observer for reflected-light observation (Epi-fluorescence) • Turn on the HBO 100 power supply switch (Fig. 59/1). • Click on the Reflected Light button and set the shutter to Open.
Page 66: Microscope Control Tool For Axio Imager.z2
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.1.2 Microscope Control Tool for Axio Imager.Z2 • Click on Locate tab in the Left tool area. Click Online if needed. • The currently set light path of the microscope is displayed.
Page 67 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Microscope settings on Axio Imager for transmitted-light observation • Set the reflector turret position to None and click the On button for transmitted light. • Pull out the push rod (Fig. 61/1) to switch the beam path of the tube in LSM position.
Page 68 Left Tool Area and Hardware Control Tools LSM 880 Fig. 61 LSM 880 with Axio Imager.Z2 • Via the Objective button, select the required objective as follows: − Open the graphical pop-up menu by clicking on the Objective button. − Click on the objective you want to select.
Page 69: Controls For Axio Examiner
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Microscope settings on Axio Imager for reflected-light observation (Epi-fluorescence) • Turn on the HBO 100 W or X-Cite power supply with switch (Fig. 61/8) • Click on the RL reflected light button. The shutter opens.
Page 70: Incubation Tool
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.1.4 Incubation Tool To have access to incubator functions to define the atmosphere and temperature, you have to expand the Incubation tool by pressing the arrow in the Incubation tool (Fig.
Page 71: Acquisition Tab
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Acquisition Tab Acquisition contains functions microscope that are needed to set up and control wide-field imaging by bright field or fluorescence, designated in this manual as wide-field (WF), laser...
Page 72: Configuration Functions
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.1 Configuration Functions load predefined acquisition configuration from the Experiment Manager field (Fig. 65) by pressing the Load button ( Fig. 65 Experiment Manager field with...
Page 73 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Select the acquisition configuration from the Parameters list by highlighting it or search for it by typing its name in the Search field. An Info menu pops up (Fig.
Page 74: Smart Setup
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.2 Smart Setup Smart Setup (Fig. 70) provides an intuitive, user-friendly interface for automatically configuring the LSM’s light path for detecting fluorescent dyes. Similar to cameras used in photography, a panel of motifs helps setting the scanning parameters for imaging with the LSM.
Page 75 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Configure your experiment The fluorescent labels used for staining the sample need to be added to the table in Configure your experiment. Dyes are added to this list from a searchable library containing almost all commercially available fluorescent labels (Fig.
Page 76 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Motifs Like in photography, common imaging conditions with typical requirements also exist in laser Fig. 73 The motifs provide presets for scanning microscopy. typical imaging tasks. The selected The Motifs in Smart Setup represent presets of motif is highlighted.
Page 77 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Proposals The imaging parameters of the Imaging Setup Tool are defined for the dyes indicated in the panel: Configure your experiment. Based upon this set of dyes used for staining the sample (see above) Smart...
Page 78 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Smart Setup displays its proposals side-by-side for optimal comparison. The proposal selected for imaging is highlighted. Key aspects of each proposal are displayed as a bar chart representation.
Page 79: Action Buttons
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.3 Action Buttons Action buttons provide control for how the image is acquired (Fig. 76). Fig. 76 Action buttons They have the following functions, regardless of the imaging mode: AF Find Focus triggers a frame z-Scan.
Page 80 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Continuous produce images from all active channels and tracks, thereby ignoring the settings for multidimensional acquisition, but taking into account the selected imaging mode. The last scanned frame will stay in the image window.
Page 81: Multidimensional Acquisition Settings
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.4 Multidimensional Acquisition Settings In the Multidimensional acquisition field (Fig. 82), the multidimensionality can be defined by checking the corresponding boxes. Fig. 82 Multidimensional input field – Z-Stack (with 10 slices), Time series (with 10 images) and...
Page 82 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Fig. 84 Multidimensional Acquisition Tool group – all possible tool tabs displayed Note that not all functions are available for all imaging modes. Only those functions that are available can be activated;...
Page 83: Setup Manager - Laser
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.5 Setup Manager – Laser Open (and close) the Laser tool (Fig. 86) (only available when the Show all tools check box is ticked) by clicking the Laser tab in the Setup Manager tool group.
Page 84 Left Tool Area and Hardware Control Tools LSM 880 Diode lasers The LSM 880 can be equipped with Diode 440 and / or Diode 405 lasers that can be used in continuous wavelength (cw) mode or in pulsed (ps) mode (Fig. 88).
Page 85 Please check the key switch at the power supply of the laser! If the failure persists contact your local Zeiss service team." -> In this case turn the key switch to 1 to be able to start the laser. Click On or Standby in the Laser tool window again.
Page 86: Setup Manager - Experiment Designer
ZEISS Left Tool Area and Hardware Control Tools LSM 880 Maximal in the drop down list next to the Power status. The maximal available laser power changes to a higher percentage value and the value is now indicated in red as well as the higher tube current. This operation mode reduces the life time of the laser and should only be used for short periods of time.
Page 87 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The active acquisition parameters include all settings of the following tools: Imaging Setup − Acquisition Mode − Channels − Focus Devices and Strategy − Z-Stack −...
Page 88 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Loops and Repetitions are set (in Show all mode) in the Loops and Repetition panel. Use the arrow to open the panel (Fig. 94). The number in the...
Page 89 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Autofocus Strategies use the center position of a Z-Stack as the reference position. For a given − Z-Stack go to the center position and if necessary, click Center within the Center tab of the Z-Stack tool once to confirm the reference.
Page 90: Setup Manager - Imaging Setup
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.7 Setup Manager – Imaging Setup The Imaging Setup tool (only available when the Show all tools check-box is ticked) allows new / existing beam path configurations to be created / edited and saved for the different imaging modes.
Page 91 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS When in Channel mode the image acquisition can either be done in one Track (all lasers and detectors active simultaneously) or in up to 4 tracks (Fig.
Page 92: Filter Cube Selection
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.7.2 Filter Cube Selection The filter cube can be selected from the reflector turret via a graphical pop-up menu when the Filter cube button is pressed (Fig.
Page 93: Stage Control
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.7.4 Stage Control If the Stage button is pressed, the Stage control panel will appear (Fig. 102). By pressing the Stop button you can interrupt any stage movement.
Page 94: Focus Control
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.7.5 Focus Control If the Focus button is pressed, the Focus control panel will appear (Fig. 103). The Z-Position display box shows the current z-position. The number can be edited by typing or using the arrowheads.
Page 95: X-Cite Control
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.7.7 X-Cite Control When the X-Cite On/Off button (only available for WF imaging mode) for reflected light of the X-Cite or HXP lamp is pressed, the control panel for the light settings is displayed (Fig.
Page 96: Ratio Imaging
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.7.9 Ratio Imaging In the Show all mode Ratio imaging becomes available (only for WF and LSM imaging modes). A click on the arrow next to Ratio provides access to the Ratio channel interface panel (Fig.
Page 97: Control Elements Of The Imaging Setup Tool
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.7.11 Control Elements of the Imaging Setup Tool This tool displays the hardware settings of the scan head (LSM tab), the beam path to image with the camera (WF tab) or the Non descanned Detectors (Non Descanned tab).
Page 98 ZEISS Left Tool Area and Hardware Control Tools LSM 880 A beam splitter is chosen according to the selected laser line(s). It separates the excitation light from the emission signal. These filters reflect the wavelength indicated and transmit all other wavelengths.
Page 99 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Activation/Deactivation of Channels • To activate a channel for imaging, check the check box next to channel icon. To deactivate the channel uncheck this box.
Page 100 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Use the slider to set the utilizable laser intensity (recommendation: start at 1 %). − The transmittance of the Acousto-Optical Tunable Filter (AOTF) changes accordingly.
Page 101 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Ratio Imaging Configuration In the Show all mode of the Imaging Setup tool two additional ratio imaging channels R1 and R2 can be defined. These channels are displayed in addition to the maximum of 8 active imaging channels.
Page 102 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Imaging in Lambda Mode (to be selected in the Imaging Setup tool). The Lambda Mode is recording the overall emission from the sample onto a wavelength-dispersive element and is imaged on a 34-channel detector.
Page 103 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS QUASAR detector settings Use the two sliders to define the spectral detection range of the QUASAR detector. Resolution: Step size can be 3.0, 4.4, 8.9, 17.8, 26.7 or 35.6 nm.
Page 104 (X-Cite 120 / HXP). It also provides access to the condenser lens aperture and filters and the tube lens. The use of this function permits the use of a Zeiss AxioCam camera (various models, see ANNEX for description) as an alternative external detector.
Page 105: Setup Imaging With Airyscan Detector
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.7.12 Setup Imaging with Airyscan Detector Acquisition, Imaging Setup, Detector Activation • To activate the Airyscan detector, tick the ChA checkbox in the beam path (Fig. 118).
Page 106 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Multitracking A multitracking acquisition is set up by using the + button (Fig. 120). In each track individual settings can be configured for the Airyscan or the internal detectors.
Page 107: Acquisition Parameter - Acquisition Mode
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.8 Acquisition Parameter – Acquisition Mode Acquisition Mode tool window, parameters for the acquisition can be set (Fig. 122). The user interface is adapted depending on the chosen Imaging mode in the Imaging Setup tool.
Page 108: Scan Mode
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.8.2 Scan Mode The mode of image acquisition (frame wise, line wise, spot wise) can be selected in the Scan Mode drop down menu. For the LSM imaging mode you can select between Frame, Line and Spot.
Page 109: Averaging And Color Depth Selection
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.8.4 Averaging and Color Depth Selection You can select the number of image frames you want to average from the Averaging Number drop down menu (Fig. 127).
Page 110: Hdr(High Dynamic Range)-Imaging
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.8.5 HDR(High Dynamic Range)-Imaging High dynamic range (HDR) creates an image with a depth of 32-bit therefore covering a far broader dynamic range than conventional images (8 to 16 bit).
Page 111: Scan Area Definition
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS In the Illumination method number of frames is automatically set to 3 (Fig. 132). The Illumination method changes the laser power Fig. 132 HDR Illumination mode (Fig.
Page 112: Acquisition Mode Functions For Lsm
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.8.7 Acquisition Mode Functions for LSM The parameters for image acquisition are set using the tool Acquisition Mode. The following scan modes, which can be selected from the drop down menu, are available: Frame −...
Page 113 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Frame Mode for LSM When the scan mode Frame is active, a frame of variable size is scanned pixel by pixel and line by line. The laser beam is moved over the specimen line by line.
Page 114 ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Select the Scan Speed from the 19 preset steps via slider or input box. Recommended: 8 for the first scan. A click on the Max button sets the maximum speed for the current zoom.
Page 115 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS In the Sum method, the intensity values of all images are added up, without a mean value being calculated. To create the image information using the Line average mode, each line (depending on the setting) is scanned 2, 4, 8 or 16 times during Scan Average, and then the average value per pixel is calculated.
Page 116 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Frame Mode for Camera When images are acquired with the camera only Frame mode is available. The following additional parameters and be set: Objective, Frame Size, Format, Bit Depth, Averaging and Offset The exposure time is set in the Channels tool.
Page 117 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Line Mode In Line mode, fluorescent or reflected light along a straight or freely definable line is displayed in the form of an intensity profile. The line is scanned pixel by pixel.
Page 118 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 If the defined free shape curve becomes too complicated or the selected Scan Speed is too high, the following message appears in the status bar: Maximum scanner acceleration exceeded! •...
Page 119: Acquisition Mode Settings For Airyscan
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.8.8 Acquisition Mode Settings for Airyscan Objective, Scan Area, Zoom The required acquisition settings for the Airyscan are set in the Acquisition Mode tool (Fig. 140).
Page 120 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Airyscan Multitrack ILEX For best synchronicity in a multitracking acquisition with the Airyscan, a so called ILEX (interleaved laser excitation) mode is available. Fig. 142...
Page 121: Acquisition Parameter - Channels
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.9 Acquisition Parameter – Channels In the Channels tool window the following options are available: − Switching, activating, deactivating and naming/saving of tracks − Defining Detector settings −...
Page 122: Defining Channels And Tracks
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.9.1 Defining Channels and Tracks The Channels tool lists all tracks in the Track Selection display area that have been defined in the Imaging Setup tool.
Page 123 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The color of a track can be set or changed selecting a LUT from the Look Up Table (LUT) drop down menu (Fig. 148). The scroll bar allows access to all predefined colors.
Page 124 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 All saved track configurations will be listed in the Load Track Configuration panel (Fig. 151). Select the appropriate one by highlighting it in the Recent section, which lists the recently used track...
Page 125 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Info window pops up (Fig. 153). Press Yes to delete the configuration, No to keep the configuration. To save a configuration press the Save button (symbolized by the disc icon).
Page 126: Ratio Channels
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.9.2 Ratio Channels The parameters for ratio metric imaging can be set when a ratio channel is selected. • Click on the button of a ratio channel (e.g.
Page 127: Using Gaasp Detectors And The Temperature Controlled Side Pmt For Photon Counting Imaging
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.9.3 Using GaAsP Detectors and the temperature controlled side PMT for Photon Counting Imaging In the LSM Imaging Setup tool activate any GaAsP detector like BiG.2, the 32 channel GaAsP Array detector or the temperature controlled side PMT (Ch2).
Page 128 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Analog (Grey Value) Display In this display, photons are transformed into grey values, which are displayed in discrete intervals in the histograms. Intervals can be calculated by: Interval = (pixel period x frequency) / (2n-1) = (pixel period x CRmax/digital gain) / (2n-1), where n = 8 for an 8 bit image and n = 12 for a 12 bit image (see Fig.
Page 129 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Counting (Raw Data) Format In this display, photons are displayed according to their number (Fig. 162). Fig. 162 Counting display in photon counting image. Settings were digital gain 1, scanning speed 8 corresponding to a pixel period or pixel dwell time 1.52 µs, 8 bit image...
Page 130: Channel Settings For Airyscan
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.9.4 Channel Settings for Airyscan The detection settings of the Airyscan can be controlled in the Channels tool (Fig. 163). The pinhole will be set automatically to a correct opening depending on the Airyscan mode.
Page 131 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Optimal pixel number and size Depending on the detection mode, the required oversampling value for the pixel number and size changes. This is adjusted automatically by using the Optimal button in the Acquisition Mode dialog (Fig.
Page 132: Acquisition Parameter - Focus
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.10 Acquisition Parameter – Focus If the Focus tool tab is expanded the Focus control tool becomes available (Fig. 168). The Z-Position display box shows the current z-position.
Page 133: Acquisition Parameter - Focus Devices And Strategy
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.11 Acquisition Parameter – Focus Devices and Strategy Focus Devices and Strategy aims to maintain focus stability in multi-dimensional acquisition experiments. This tool is typically employed to compensate for Z-...
Page 134 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Off mode If Off is selected, autofocus is inactive and will not be used. If a multi-track acquisition includes a track that employs a camera for data capture, the autofocus mode is automatically set to Off in Focus Devices and Strategy.
Page 135 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Find offset searches for a reflective surface (the focus reference plane) starting at the Z-level of the imaging plane. Next, it copies the increment between the Z-levels of the imaging plane and the focus reference into the Offset spin box.
Page 136 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Here, the Autofocus every n Timepoint check box is checked and a value of 3 entered in the associated spin box. A focusing event will be executed before the first acquisition and after the completion of three acquisition cycles.
Page 137: Acquisition Parameter - Stage
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.12 Acquisition Parameter – Stage The following software description applies to systems which are equipped with a motorized scanning stage. If the Stage tool window is expanded the Stage control tool becomes available (Fig.
Page 138: Acquisition Parameter - Shuttle And Find
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.13 Acquisition Parameter – Shuttle and Find Shuttle and Find allows calibration and imaging of sample positions in laser scanning microscopes (LSM) for reproducible repositioning after transfer to scanning electron microscopes (SEM).
Page 139 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Mounting of cover glasses or TEM grids • Place the specimen (sample carrier) in the suitable sample holder and fix it: Life Science Cover Glass 22x22: −...
Page 140 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Calibration The calibration markers consist of one small and a large L-shape marker. The bigger marker is used for coarse orientation, whereas the smaller marker is used for the calibration.
Page 141 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Threshold: High – Low: A low threshold for marker detection is used when the dimensions of the correlative L markers cannot be recognized precisely, e.g. when the sample holder is slightly filthy.
Page 142 ZEISS Left Tool Area and Hardware Control Tools LSM 880 • If the Automatic Movement to next Position is activated, the stage will move automatically to the next marker (Position 2) after clicking Accept. Otherwise, the marker 2 has to be found manually.
Page 143 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Image acquisition and saving • Acquire images of your sample as you are used • For saving (Fig. 183) the images together with coordinates and defined regions of interest or points of interest use the .czi file format.
Page 144: Acquisition Parameter - Regions
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.14 Acquisition Parameter – Regions The Regions tool tab is only available, if the Regions checkbox ticked Multidimensional acquisition field. If the tool bar is expanded, the Regions tool becomes available (Fig.
Page 145 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The following tools are available in the Tool selection field: Selects an existing region in the image. The outline of the region is marked with small rectangles to show it is active.
Page 146 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 When highlighting a ROI in the list, the numerical parameters for Center X, Center Y, Width and Height in pixels will be shown in Region feature field.
Page 147: Multidimensional Acquisition - Z-Stack
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS In this case the scanner movement will be restricted to the bleach region zooming in onto this region. This may result in a less accurate positioning of the region as the definition of the region has been made in a different zoom in the image.
Page 148: Performing A Z-Stack Using First/Last Mode
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.15.1 Performing a Z-Stack Using First/Last Mode Press the First/Last mode button to open the First/Last definition panel. Press the Continuous action button and focus onto the specimen.
Page 149 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Position (µm) input box indicates the current position of the focus, no matter if the focus drive or a Z-Piezo stage insert was used for positioning. The focus is changed in small steps when clicking the arrow keys.
Page 150: Performing A Z-Stack Using Center Mode
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.15.2 Performing a Z-Stack Using Center Mode Press the Center mode button to open the Center definition panel (Fig. 194). This mode allows setting the Range of the Z-Stack in relation to the current Focus Position which is indicated as Offset.
Page 151 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Pressing the Center button will define the current focus position (as defined in the Position (µm) input box) as the center position of the stack. After pressing Center the whole range will move by the difference between of the previous center position and the new one.
Page 152: Performing A Z-Stack Using A Z-Piezo
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.15.3 Performing a Z-Stack Using a Z-Piezo Z-Piezo allows Z-Stacks acquired considerably quicker compared to the focus drive of the microscope stand. The speed advantage depends on the total travel range in Z and the Fig.
Page 153: Optimize Sectioning And Step
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.15.4 Optimize Sectioning and Step Expand the Optimize Sectioning and Step field by pressing the triangle (Fig. 196). The Optimize Sectioning and Step function helps to set the optimal Interval.
Page 154: Correction
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.15.5 Correction Expand the Correction field by pressing the Refractive Index triangle (Fig. 198). Correction input box allows you to consider the different refractive indices between the immersion Fig.
Page 155: Z-Stack Settings For Airyscan
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.15.6 Z-Stack Settings for Airyscan In case a 3-dimensional acquisition is required, a Z-Stack (Fig. 200) can be defined the same way as when using the internal channels.
Page 156: Multidimensional Acquisition - Bleaching
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.16 Multidimensional Acquisition – Bleaching When the Bleaching tool tab is expanded the Bleaching tool becomes available (Fig. 202). The Bleaching tool is only available in LSM Imaging mode.
Page 157 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS If a region is used to define a position for bleaching within the sample, the bleach process can be accelerated by checking the box next to Zoom Bleach (fast, less accurate). In this case the scanner movement will be restricted to the bleach region zooming in onto this region.
Page 158: Multidimensional Acquisition - Tile Scan
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.17 Multidimensional Acquisition – Tile Scan When the Tile Scan tool tab is expanded the Tile Scan tool becomes available (Fig. 204). This function permits a frame to be created as an overview image of the specimen.
Page 159 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Tick the checkbox to activate online stitching. After ticking the checkbox the Threshold input box becomes available. Enter the desired threshold in %. By pressing Add you can mark stage positions, which will be listed in the Marked position field in terms of Number as well as x [µm] and y [µm]...
Page 160: Scan Overview Image For Tiling
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Marked positions are listed up. This list can be saved and loaded. Fig. 209 Tile Scan: Marked positions 5.2.18 Scan Overview Image for Tiling Pressing the Scan overview Image… button opens the Scan overview Image interactive window (Fig.
Page 161: Multidimensional Acquisition - Time Series
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.19 Multidimensional Acquisition – Time Series When the Time Series tool tab is expanded the Time Series tool becomes available (Fig. 211). In the Time Series tool the parameters for the...
Page 162 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The trigger can be selected from the Trigger In pull down menu. Up to 4 triggers are available. The trigger has to be assigned in the firmware of the system. If no trigger is set the highlighted delay time will be effective for the time series acquisition.
Page 163: Marker
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Select timer configuration from Configurations list by highlighting it or search for it by typing its name in the Search field. The Info window pops up (Fig. 216).
Page 164 ZEISS Left Tool Area and Hardware Control Tools LSM 880 All saved marker configurations will be listed in the Load configuration… panel (Fig. 220). Select the appropriate one by highlighting it in the Recent section, which lists the recently used marker configurations or from the Configurations section, where all saved marker configurations are displayed.
Page 165: Start
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Info window pops up (Fig. 222). Press Yes to delete the configuration, No to keep the configuration. To save a marker configuration press the Save button (symbolized by the disc icon).
Page 166 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 If Trigger is selected the Trigger In drop down menu becomes available (Fig. 226) from which up to 4 triggers can be selected. After pressing Start...
Page 167: End
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.19.4 Expand the End field by clicking the triangle (Fig. 230). In the Mode drop down menu (Fig. 231) Fig. 230 End field you can select the way the acquisition is triggered.
Page 168: Multidimensional Acquisition - Positions
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.20 Multidimensional Acquisition – Positions When the Positions tool tab is expanded the Positions tool becomes available (Fig. 234). This function comprises two main applications.
Page 169: Sample Carrier
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.20.2 Sample Carrier Press the Sample Carrier button to obtain the Sample Carrier control panel (Fig. 236). The last used sample carrier will be displayed in the Sample Carrier display area.
Page 170 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 All saved marker configurations will be listed in the Load configuration… panel (Fig. 239). Select the appropriate one by highlighting it in the Recent section, which lists the recently used marker configurations or from the Configurations section, where all saved carrier configurations are displayed.
Page 171 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Select the carrier configuration from the Configurations list by highlighting it or search for it by typing its name in the Search field. The Info window pops up Fig. 241).
Page 172: Setting Individual Offset Values For Positions In Position List Mode
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.20.3 Setting Individual Offset Values for Positions in Position List Mode The Focus Devices and Strategy tool contains the controls for maintaining focus stability when using the Positions tool (e.g.
Page 173: Scan Overview Image
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.20.5 Scan Overview Image Pressing the Scan overview Image… button opens the Scan overview Image interactive window (Fig. 245). Here you can define the number of Tiles by using the Horizontal and Vertical Input boxes (the extension of the scanned area will be displayed in Pixel and Size (µm)
Page 174: Multidimensional Acquisition - Auto Save
This option is not available for the Carl Zeiss Image (CZI) file format. If the Separate Files checkbox is not ticked, ticking the respective Sub-directory checkbox is of no consequence.
Page 175: Image Optimization
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.23 Image Optimization This section describes an example how to acquire an image, using an excitation wavelength of 561 nm and a fluorescence emission range above 570 nm.
Page 176 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Activate channel 1 via the check box. You may want to assign a different color to the channel which will be taken over for the initial display of the image.
Page 177: Defining The Scan Parameters
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.2.23.2 Defining the Scan Parameters • Open the Acquisition Mode tool (Fig. 252). • Select Frame mode and a Frame Size of 512 x 512 to start with.
Page 178: How To Optimize Image Quality
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.2.23.4 How to Optimize Image Quality As a rule, the first scanned image is not ideal, since the settings of the laser and therefore also the photomultiplier might not match the light output.
Page 179: How To Optimize Imaging With Airyscan
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS A similar procedure applies for image optimization if more than one channel is used and simultaneous image acquisition is performed. Make sure the suitable lasers are switched on and the channels are activated.
Page 180 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Raw data sum image, Range Indicator (Fig. 257) In order to fine tune the detection, the raw sum image (source image, left) of the Airyscan should be observed carefully.
Page 181: Fcs Tab
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS FCS Tab The major functions can be selected from the FCS tool group (Fig. 258) in the FCS tab. To open saved files click the File button in the menu bar.
Page 182 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Start Experiment Triggers measurements according to the settings at all defined positions. action button Stop action button Will end an active measurement. Data will be available to store up to the end point.
Page 183: Setup - Laser
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.1 Setup – Laser Open the Laser tool by clicking the Laser tab in the Setup Manager tool group (Fig. 259). Close the tool box by clicking again.
Page 184 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Saving and deleting beam paths You can save a beam path by typing in a memorable name intothe drop down menu selection box and pressing the Save button (floppy disc icon). If you save a beam path under the existing name you will be prompted to confirm overwriting the old configuration.
Page 185: Light Path For 2 Channel Module (Big.2 On Dc Out Port)
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.2.1 Light Path for 2 Channel Module (BiG.2 on DC out port) BiG.2 has two GaAsP detectors that can be used simultaneously to acquire two autocorrelation functions (AC) per each channel.
Page 186 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The icons are inactive, as the EFs are part of the filter cube together with the SBS filter, which has to be replaced manually. The filter are indicated to SW within the Filters Tool window (Maintain tab). Note, that if the filter is exchanged manually and the database is not updated, the old filter names will stay.
Page 187: Ndd Light Path (Big.2 As Ndd)
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.2.2 NDD Light Path (BiG.2 as NDD) BiG.2 can be attached to the transmitted (NDD-T) and/or reflected (NDD-R) position (not more than two BiG.2 modules in can be used on one system...
Page 188 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The icons are not displayed, because the EFs are part of the filter cube together with the SBS filter, which has to be replaced manually. The filter are indicated to SW within the Filters Tool window (Maintain tab).
Page 189: Light Path For 32 Channel Module
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.2.3 Light Path for 32 Channel Module The 32 Module consists of an array of 32 GaAsP detectors plus two side PMTs. The side PMT of the longer wavelength region (Pmt2) can be used together with the GaAsP array detector for FCS.
Page 190: Selection Of Laser Lines
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.2.4 Selection of Laser Lines • Press the Laser button to display the laser control expansion submenu (Fig. 265). Alternatively you have access to the Laser Lines in the Acquisition tab.
Page 191 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Lasers functionalities for laser control are also available in the Acquisition tab (Fig. 268). Only that all visible and invisible lasers are displayed together (als applies if Show All mode is not set in the Light Path tool window).
Page 192: Pinhole Adjustment
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.2.5 Pinhole Adjustment The Adjust Pinhole panel allows you to set the pinhole diameter and to automatically align the pinhole position in x and y.
Page 193 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The pinhole is adjusted using a dye solution. The general approach is to move the pinhole cyclically in x and y until the intensity maximum is found. For each excitation wavelength a suitable dye must be used.
Page 194 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Fig. 270 Automatic Pinhole Adjustment in x coarse (left) and x fine (right) • You can also alter the position by typing into the input boxes or using the sliders or scroll arrows. If both coordinates are changed, the system will regard this as a new alignment.
Page 195 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Procedure to adjust Pinholes • Mount a carrier with a suitable dye solution and orient the sample stage. Select the test sample. • Select the beam path or the method with attached beam path you want to use.
Page 196: Setup - Acquisition
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.3 Setup – Acquisition Measure Time [s] The Measure Time (Fig. 271) is the period of one measurement. rule thumb measurement time should be about 1000 x the diffusion time of a molecule under study.
Page 197 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS • Set the Measure Time. If you do not have any idea about the behavior of your sample, start with a measurement time of about 10 seconds. Depending on the signal-to-noise ratio of your correlation curve, decrease or increase the measurement time.
Page 198: Setup - Fit
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.4 Setup – Fit The Fit tool allows you to load a model to be used for the measurement as well as to define new models.
Page 199 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS − Upper Limit: defines the upper limit value tolerated from a fit; if this value is exceeded, the fit is rejected and another (global) minimum is searched for. The default value depends on the parameter.
Page 200: Defining A New Model
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.4.1 Defining a New Model If the Define button is pressed, the Define Model panel will open. It is used to define model equations to which measured data can be fitted.
Page 201: Correlation Options
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.4.2 Correlation Options In correlation you can assemble a model with predefined terms. • Press the Correlation tab to activate the options for assembling a correlation model (Fig.
Page 202: Model Equations
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.4.3 Model Equations In the following, the mathematical equations of the correlation functions and the fit equations will be more detailed. The acquired correlation functions must be fitted to models in order to retrieve meaningful parameters.
Page 203 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Definition of the cross-correlation function The formalism for the cross-correlation function is identical to the auto-correlation function, with the exception that the signal in one channel is not compared to itself, but to a signal in a second channel.
Page 204 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 A. Amplitudes The Amplitude of the correlation function is influenced by the offset, background and the number of particles in dependence of the geometric factor. The amplitude is also influenced by the process of correlation.
Page 205 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Amplitude A γ ( − (5a) where γ is the geometric factor accounting for the point spread function (PSF) and N the mean number of particles.
Page 206 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 The following relations exist: ω (5e) for 1 photon excitation ⋅ ω (5f) for 2 photon excitation ⋅ ω (5g) ω Equations 5e or 5f, dependent on the excitation source, can be used to retrieve ω...
Page 207 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS B. Anti-bunching terms Anti-bunching is the phenomenon that a molecule cannot produce emitted photons as long as it stays in the excited state. Hence during the transition time required to drop back to the ground state, which...
Page 208 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 and t is a fixed parameter and must be user defined; κ are fit parameters; k is either a fit parameter or can be fixed.
Page 209 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS C. Bunching terms Bunching is the phenomenon of a burst of photons during a certain time interval, the duration of which is determined by photo-physical processes including triplet, blinking, flickering and protonation. These terms are exponential decay functions.
Page 210 ZEISS Left Tool Area and Hardware Control Tools LSM 880 Blinking is based on the phenomenon that the electron distribution over conjugated systems can change in dependence on the local environment, for example changes in the pH, which will lead to molecules in a bright and dim or dark state.
Page 211 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS C.4 Independent triplet and blinking In this case the terms are just representatives for two dependent bunching terms that are linked by multiplication. Note that the triplet fraction, if present, could be fitted to either of the terms.
Page 212 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 C.6 Double stretched exponential - bunching This is a double exponential function, where the exponentials are added. κ κ − ⋅ − ⋅ − ⋅...
Page 213 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS D. Diffusion terms Diffusion is driven by Brownian motion. We can distinguish translational, rotational and flow diffusion. D.1 Rotational diffusion In the most general form, rotation can be described as the sum of 5 exponential terms −...
Page 214 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 − − ⋅ ⋅ ⋅ normalized (8f) − frequency factors and t and t with R being the amplitude, c and c relative amplitudes, r and r rotational diffusion times.
Page 215 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS D.2 Translational diffusion In its general form, translational diffusion is defined as:           Φ ∑ ∑...
Page 216 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 are fitted parameters. They can be converted to diffusion coefficients D using formulas 5e or 5f. The values, but in this case the lateral radius ω...
Page 217 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Note that the brightness contributes as the square to the correlation function, in other words a double as bright molecule will contribute 4 fold more. Therefore, the fitted number of molecules must be corrected to obtain the real number N of diffusing particles;...
Page 218 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 D.3 Flow Flow signifies active transport either via cytoplasmic movement or directed transport. If flow occurs in the absence of translational diffusion, the term is defined as follows: ...
Page 219: Definition Of A Model
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.4.4 Definition of a Model • To define a model press the Term tab of the term you want to be part of your final correlation equation.
Page 220 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Background • You can set an offset by setting a value in the Background selection box (Fig. 277). Either type in a value or use the arrows.
Page 221 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Antibunching You can select the anti-bunching term in its normalized form by checking the Normalized check box or in its non-normalized form by unchecking the box (Fig. 279).
Page 222 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Triplet The triplet represents bunching terms that are exponential decay functions. You can select the triplet term in their normalized form by checking the Normalized check box or in their non-normalized form by unchecking the box (Fig.
Page 223 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Rotation You can choose between normalized and non- normalized rotation functions by selecting / dese- lecting the Normalized check box (Fig. 283). You can select the Dependent antibunching, rotation and translation by checking the respective box.
Page 224 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 You can choose between symmetric rotation (1 exponential term) and asymmetric rotation (double exponential term) by selecting / deselecting the Asymmetric check box (Fig. 285).
Page 225 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Translation You have the following possibilities for setting parameters (Fig. 286): • You can either fit to fractions (used normally when no brightness differences are observed...
Page 226 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Flow You can determine, if you want to fit to the diffusion time or to the velocity directly by un- ticking or ticking the Velocity check box (Fig.
Page 227 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Stretched exponential You can select between bunching and anti- bunching terms by pressing the Bunching or Antibunching buttons, respectively (Fig. 288). You can select between 1 (mono exponential) or 2 (double exponential) stretched exponential terms by pressing the Components 1 or 2 buttons.
Page 228: Pch
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.4.5 In the PCH (Photon Counting Histogram) panel you can determine concentrations and the molecular brightness of molecules. • Click on the PCH tab to open the PCH control panel (Fig.
Page 229: Formula
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.4.6 Formula The Formula panel allows you to define your own fit equation. • Press the Formula tab to open the Formula panel (Fig. 290). Click any other tab to leave the panel.
Page 230: Setup - Correlator
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.5 Setup – Correlator In the Correlator tool you can define the fit and acquisition parameters for the measurement. • Click on the Correlation tab to open the Correlation panel (Fig.
Page 231 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS together and normalized. The normalization result is defined as: (result x received clocks)/(pulse for auto-correlation or (result x received clocks)/(pulseA x pulseB) for cross-correlation. The received clocks counted equal 4x the number of correlation intervals, which are defined by the measurement time.
Page 232 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 In PCH panel the binning time used for the photo counting histogram can be selected. • Click on the PCH tab to open the PCH panel (Fig.
Page 233 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Please note that the cut off count rate is defined as a value exceeding the average count rate during a certain measurement period (bin window) by a certain percentage. Thus, the consecutive...
Page 234: Navigation - Stage
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.6 Navigation – Stage • Click on the Stage tool tab to open the Stage panel (Fig. 296). The Compass Rose allows you to move the...
Page 235: Navigation - Sample Carrier
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.7 Navigation – Sample Carrier The Sample carrier tool allows the definition and orientation of a sample carrier, so that single wells can be approached automatically.
Page 236 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 In order to delete a configuration, press the Delete button (cross symbol). This will open the Delete configuration… window (Fig. 302). Select the configuration you want to delete by clicking on its name.
Page 237: Navigation - Focus
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.8 Navigation – Focus The function of the Focus tool allows you to position the focus in Z direction. • Press the Focus tool tab to open the Focus tool (Fig.
Page 238 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Proceed as follows: • Make sure that the front lens of the water immersion objective is wetted by a drop of water. Use fluorescence-free double-distilled...
Page 239 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS • Set up scan mode in the LSM Acquisition Mode tool (Fig. 306). Choose Line scan, highest Scan speed, 512 pixels and Zoom 1 for convenience.
Page 240 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Press Continuous to start a continuous scan. Press the Line Scan tab (Fig. 308). • The diagram (Fig. 309) shows a line with a...
Page 241 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS • Continue moving upwards slowly. The line position will reach its maximum (Fig. 311). Fig. 311 Line diagram at lower cover slip surface • Having slowly moved over a short distance the...
Page 242: Multidimensional Acquisition Tools - Time Series
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.9 Multidimensional Acquisition Tools – Time Series The Time Series tool is only available if the Time Series box in the tab activation area is checked (Fig.
Page 243 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS If the kinetic will last longer than one day, a warning is displayed (Fig. 317). Click Ok to continue. Define the following variables: Time delay between consecutive data points = pi...
Page 244: Multidimensional Acquisition Tools - Positions
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.10 Multidimensional Acquisition Tools – Positions In the Positions tool you can select the carrier and the sample or laser position by the stage or by the scanners.
Page 245 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS If the XY Stage button is activated, all positioning will be done via the scanning stage. This mode corresponds to the Sample Carrier mode, if Single Position is selected. Regardless which well is selected, in the result table of the FCS results window the position is indicated as "1".
Page 246 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Sample carrier The Sample Carrier is used to approach a single well or multiple wells sequentially in an automatic mode. • Click on the Sample Carrier tab to open the Sample Carrier panel (Fig. 321).
Page 247 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS • Click on the corresponding chamber in the Sample Carrier window (Fig. 321). − This chamber will be flashing up briefly. The stage is now oriented. Note that there is no further indication that the stage was oriented.
Page 248 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Single Position Only a single well can be activated. The scanning stage will automatically move to the activated well without the need to trigger a measurement.
Page 249 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Multiple positions • Select from the Working Direction drop down menu an entry other than Single position. • Select as many individual well positions as required (all are selectable) by just clicking on them. All selected wells will be highlighted in blue.
Page 250 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Move the cursor to the desired position in the scan image (drag and drop). Press the Add button to add the position marked with the crossline to the Positions list (Fig.
Page 251 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The X-, Y- and Z-coordinates of the selected positions are only stored in the Positions table and not in the scanned image (stack). Accordingly, all the marked positions in all Z-planes are displayed in the scanned image (Z-Stack), no matter in which Z-plane they are.
Page 252 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 To compensate for scan zoom and scan speed go to the Acquisition Mode too of the LSM Online Acquisition tool group. Use a sample with high contrast, like "Convolaria", and focus.
Page 253: Navigation - Incubation
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.11 Navigation – Incubation Open the Incubator tool by clicking the Incubator tab in the Navigation tool group (Fig. 331). You will have access dependent on the attached equipment to −...
Page 254: Multidimensional Acquisition Tools - Information On Experiment
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.12 Multidimensional Acquisition Tools – Information on Experiment • Click on the Information on Experiment tab to display a detailed description of the highlighted method...
Page 255: Action Buttons For Fcs
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.13 Action Buttons for FCS Action buttons used trigger measurements and to adjust the measurement conditions. The following buttons are available (Fig. 333): − New: opens up a new Image FCS Diagram within a container.
Page 256: Method Tool
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.14 Method Tool The Method tool allows to store (floppy disc button), load (open file button) and to delete (cross button) methods. Fig. 334 Save Method panel...
Page 257 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Loading a method The Load button (open file symbol) will allow you to load a pre-defined method. The configurations will be displayed; however, hardware settings change only if a measurement is triggered.
Page 258 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Deleting a method The Delete button (cross symbol) allows you to delete a method from the database. • Click on the Delete button. A Delete configuration…...
Page 259: Activation Tab Area
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.15 Activation Tab Area In the activation area you can select certain tabs to be displayed in the tool area or no by just checking Fig.
Page 260: Count Rate
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.17 Count Rate • Click the Count rate button to open the Count rate window (Fig. 339). Display Select one of the display modes Count rate,...
Page 261 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS • Click the CPM button to open the counts per molecule per second (CPM) (Fig. 342). This value is constantly updated and calculated each 0.5 ms if Averaging is switched Off. The blue bar indicates the extend of the CPM graphically.
Page 262: X-Y-Z Scan
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.18 X-Y-Z Scan The X-Y-Z Scan panel allows you to perform scans in the x, y and z directions and combinations thereof. Especially in the z direction, it helps to position the focus to the correct position, e.g.
Page 263 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Please note, that x and y scans are performed by the stage regardless if the Scanner button in the Measurement tool group is pressed or not. Z Scans performed nosepiece.
Page 264 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The data of the Count rate scan window can either be copied to the clipboard or written to a text file. A click in the Count rate scan window with the right mouse button will open the context menu for line or two dimensional scans.
Page 265: Snap
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Manual Focusing on the Membrane • Acquire an image of the cell. • Place the focus above the cell and scan continuously. • Focus slowly down until you see a fluorescence signal.
Page 266: Taking Fcs Measurements
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.21 Taking FCS Measurements • Click on the New document button to open a new FCS Diagram in an Image Container. New diagrams per session will be numbered starting with 0 (Fig. 347).
Page 267 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Fig. 348 Correlation tab Fig. 349 Table tab The FCS diagram contains five layers, which can be selected by pressing the corresponding tab: − Correlation: Displays the count race trace, correlation function, photon counting histogram and pulse density histogram.
Page 268 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Pressing New will open an empty FCS diagram. To generate data in the FCS Diagram you have to trigger a measurement by pressing the Snap or Start Experiment button.
Page 269: Start And End Of A Measurement
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.21.1 Start and End of a Measurement Fig. 351 FCS Correlation window during data acquisition After setting the parameters the measurement can be run. • Click on the Snap or Start Experiment action button to start the measurement and data analysis process.
Page 270 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Positioning in solution • Align a carrier in the Carrier Position tool. Focus into the solution. • Go to the Measure tool and the Acquisition panel. Choose Current Position or Sample Carrier and select the well(s) for the measurement.
Page 271 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Microscope Control tool contains the following functions: HAL On/Off button for transmitted light: If the button is pressed, the control panel appears and the shutter automatically opens (Fig. 353). If you press the On button, the light intensity of the HAL lamp can be varied via the input box or slider.
Page 272 ZEISS Left Tool Area and Hardware Control Tools LSM 880 Home and Load positions can also be accessed from the z-position slider by pressing the H or L buttons. The z-position can be altered by grapping and sliding the plane or by using the arrow slider. The plane shows the actual z-position.
Page 273: Loading Stored Data Files
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.21.2 Loading Stored Data Files To load stored data files you can use the File menu from the main toolbar (Fig. 356). • Go to File and than choose Open. This will...
Page 274: Fcs Correlation Diagram
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 The Image FCS Diagram consists of four data layers: − FCS Correlation − FCS Coincidence − FCS Fit − FCS Print Each layer will display the data as they were stored last. As default, the first layer FCS Correlation will be displayed.
Page 275 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Fig. 359 Image Container with FCS Diagram containing loaded data and FCS Correlation selected The Result Table below the diagrams displays the measuring results. The width of the columns can be changed by moving the border lines.
Page 276 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Selection of lines and properties of the table can be defined by pressing the right mouse button, when the cursor is within the table. This will open the Result Table context menu (Fig.
Page 277: Image View Controls - Correlation Tab
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Color assignment to FCS curves • Select those FCS curves in the result table with the right mouse button and pressing the Ctrl key, for which you want to assign a new color.
Page 278 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Reuse With a click on the Reuse button, the configuration used to record the selected measurement will be loaded. Reload With Reload the current active measurement will be reopened.
Page 279: Image View Controls - Table Tab
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.21.5 Image View Controls – Table Tab The Table tab (Fig. 365) chosen from the Option tool area displays several options: Fig. 365 Table tab −...
Page 280 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Count rate trace In the Count Rate diagram the count rate(s) (CR) in kHz is plotted vs. running time. If a cross correlation set-up is used, the count rate trace for each channel is displayed. If you have set an electronic dust filter in the Measurement tool in Processing, loaded raw data files with an electronic dust filter activated or placed cut limits manually the cut off regions will be displayed as matted areas.
Page 281 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Correlation G(t) The diagram shows the correlation functions for each activated channel. The zoom function can be used to display certain diagram areas of interest in an enlarged form. Use the left mouse button to click on the margin of the area of interest in the diagram, keep the mouse button pressed and draw a rectangular above the area of interest in any required direction.
Page 282 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Fig. 368 The Correlation Curve window in normal (upper graph) and normalized (lower graph) display 000000-2071-464 10/2014 V_01...
Page 283 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Pulse distance histogram This diagram shows the frequency plotted against the times elapsed between two subsequent pulses (or: photons recorded from the detector). To obtain this histogram, the time distance between two photons is evaluated from the raw data.
Page 284: Saving Correlation Results
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Choosing Abscissa logarithmic or Ordinate logarithmic will display the abscissa or ordinate in a logarithmic scaling. • The line width allows you to select the thickness of the graph. 6 settings are possible.
Page 285 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Export With Export, the measured data will be exported in a ConfoCor 1-compatible format and can be handled using the FCS Access Fit program. In the Export window you can enter a name in the File name field and a type in the Data type field.
Page 286: Coincidence
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.21.7 Coincidence The coincidence analysis generates a two-dimensional color-coded count rate histogram (Fig. 374) in this diagram. During the measurement the count rates in channel 1 (red) and channel 2 (blue) are registered in small time windows and the respective frequencies are calculated.
Page 287: Fit
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.3.21.8 • Click on the Fit tab to activate the Fit display mode (Fig. 375). In general this menu allows you to work with already measured and probably also fitted correlation curves.
Page 288 ZEISS Left Tool Area and Hardware Control Tools LSM 880 For fitting to a PCH model the upper graph (Fig. 376) shows the PCH Fit diagram curves (frequency against count rate per bin) with the overlaid fit graph. If an experiment is loaded for which no fit has been conducted yet, only the measured curve is shown and no fit curve.
Page 289 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS • Click on the diagram with the right mouse button to open the context menu for the Fit Correlation (Fig. 377) respectively Fit PCH diagrams (Fig. 378).
Page 290 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Select Normalize to display the correlation curves in a normalized form (Fig. 379). The amplitude of the diffusion contribution will be normalized to 2, not the total amplitude, if other term like triplet state were used in their normalized form.
Page 291 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The context menu of the Fit deviation diagram (Fig. 380) can be used to reset the zoom to its original value, copy the data, and adjust the display.
Page 292 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Lower and upper limits are only displayed if selected. To select, click with the right mouse button into the result table to open the context menu (Fig. 382).
Page 293 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Channel display field The Channel display field shows the active channel (selected name and color), for which the Fit All button applies. It is the last selected channel. If for example, you have selected rows in the Result table belonging to channel 1, channel 2 and a cross correlation channel, the last selected channel will be displayed and if you press Fit all selected rows will be updated.
Page 294 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The global link status of a parameter is displayed in the Link column. By clicking with the left mouse button in the respective field, a button tab becomes available. Use the arrow head to open the drop down menu.
Page 295 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS State display box The State display box (Fig. 385) above the Fit action buttons informs you about the fitting conditions. Whenever some inconsistencies are present, e.g having to parameters free that depend on each other, like the geometric factor and the number of molecules, the system gives a warning about the mistake.
Page 296: Print
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.21.9 Print Click on the Print tab to activate the Print Diagrams display mode (Fig. 387). The Print Diagrams window shows an overview of the results presented in tables and / or graphs, and relevant measuring parameters.
Page 297 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Fig. 387 Print panel 10/2014 V_01 000000-2071-464...
Page 298: 5.3.21.10 Information
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.3.21.10 Information Fig. 388 Information panel The information panel (Fig. 388) contains three blocks: Name, Comment and Acquisition. Names: This is an entry box, where you can input the name of the experimental set up.
Page 299: Processing Tab
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Processing Tab 5.4.1 General Structure of the Processing Tab Click on the Processing tab to have access to the processing functions. The list of available Processing tool will be displayed (Fig.
Page 300 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 All processing tools can be used with stored and reloaded images or with newly acquired images (that need not be stored before processing). Please note that for PALM images, although stored in a .czi format, only the PALM and Channel Alignment Processing tools are available, which can recognize tables.
Page 301 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Processing tool tab (Fig. 390) is structured into different fields. The top field or header (just beneath the Main tool tab area) contains the Apply button. The button is only active when an image is selected.
Page 302 ZEISS Left Tool Area and Hardware Control Tools LSM 880 Processing functions do not contain additional options and the field remains empty. Only those functions will be displayed that match the dimensions of the image parameters and that can be applied to the loaded image.
Page 303: Processing - Airyscan Processing
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.2 Processing – Airyscan Processing Licensing Instrument standard  optional  LSM ELYRA Lightsheet Z.1 Tool accepts Z-Stacks and time series and combinations of both. See full description of tool for details.
Page 304 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Manual • For manual processing, deactivate the Auto checkbox (Fig. 394). • In the preview window you can check again whether the right image type is selected.
Page 305 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Batch • For batch processing press the Run Batch button (Fig. 396) and select the respective directory and files from the file selection window which opens at the top.
Page 306: Processing - Maximum Intensity Projection
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.3 Processing – Maximum Intensity Projection Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts Z-Stacks and time series (incl. combinations with several other dimensions).
Page 307: Processing - Color-Coded Projection
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.4 Processing – Color-coded Projection Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts Z-stacks and time series (incl. combinations with several other dimensions).
Page 308: Processing - Image Calculator
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.5 Processing – Image Calculator Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts all types of image data recorded with the listed instruments. See full description of tool for details.
Page 309 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS For an overview of the available more advanced operators, please refer to the formula panel that is shown in a pop-up window after clicking the Operators…...
Page 310: Processing - Average
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.6 Processing – Average Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts all types of image data recorded with the listed instruments. See full description of tool for details.
Page 311: Processing - Filter
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.7 Processing – Filter Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts all types of image data recorded with the listed instruments. It is generally advisable to perform certain processing steps (e.g.
Page 312 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 The effect of the filter increases with the matrix size. This matrix / kernel size can be set for each dimension with the three sliders Kernel size X,...
Page 313 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Band Filter With the band filter (Fig. 407), the original image is filtered with a "band blocker" filter. It filters out a certain frequency band with the position set by Center, width set by Band Width and a steepness of the edges set by Strength.
Page 314: Processing - Linear Unmixing
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.8 Processing – Linear Unmixing Licensing Instrument  standard optional  LSM ELYRA Lightsheet Z.1 Tool accepts lambda stacks and multi-channel image data (single images and multi- dimensional image data).
Page 315 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS • Pressing the Select button loads the active image from the Image Display and displays it as Input Image. Only Lambda stacks (only available in LSM imaging modes) or multi-channel images can be loaded.
Page 316 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Select the sample-specific fluorescence dye reference spectra from the Spectra Database (Fig. 411). • Assign appropriate colors fluorescence channels by clicking on the color flag and choose from the available lookup tables (LUTs) •...
Page 317 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Weighted unmixing: When this option is checked, spectral channels with high noise contribute less to the unmixing result. This option includes a statistical analysis of the signal-related (Poisson-) noise and weighs the respective contribution for the fitting with the combination of reference spectra to the experimental data.
Page 318 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Multichannel Unmixing: When this option is chosen. the unmixing algorithm is applied to a multi-channel image (up to 10 channels) without the use of reference spectra.
Page 319: Processing - Ion Concentration
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.9 Processing – Ion Concentration Licensing Instrument standard  optional  LSM ELYRA Lightsheet Z.1 Tool accepts single-channel or multi-channel, multi-dimensional data (typically time series data) of samples labeled with fluorescent ion indicators. Ion Concentration was developed for analyzing LSM data.
Page 320 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Spin boxes to set the Min and Max in the Output Image Image scaling Pull down menus for To set three different calibration options, according to the dyes (single wavelength, vs ratiometric), the calibration method and if the calibration is carried out in vitro/situ.
Page 321: Single Wavelength Dyes - Offline Calibration
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.9.1 Single Wavelength Dyes – Offline Calibration • Subtract background/ autofluorescence image from raw images to obtain better raw data to start with (Fig. 417).
Page 322: Ratiometric Dyes
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.9.2 Ratiometric Dyes • Fura-2, Indo-, SNARF, Cameleon, Ratiometric Pericam, Phluorin. • Display fluorescence ratio R over time • Display fluorescence ratio R corrected for background/autofluorescence over time •...
Page 323 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Ratiometric Dyes - Calibration (Fig. 419) • Subtract background/autofluorescence images from raw images to obtain )/(F korr 1Background 2Background when calibration reference is not obtained with the experimental sample (in situ).
Page 324 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Ratiometric Dyes Equation Calibration (Grynkiewicz) (Fig. 420) Fura-2, Indo-1, (dissociation constant) taken from literature : derived from ion-free state of the dye (e.g. 0 R max: derived from ion-bound state of the dye (e.g.
Page 325: Processing - Correlation
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.10 Processing – Correlation Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts potentially all types of image files. See full description of tool for details.
Page 326: Processing - Modify Series
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.11 Processing – Modify Series Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts single image files (for concatenation) and multi-dimensional image series (typically time series and Z-stacks).
Page 327: Processing - Hdr-Imaging
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.12 Processing – HDR-imaging Licensing Instrument standard  optional  LSM ELYRA Lightsheet Z.1 " " Tool accepts image data recorded with the optional feature of the acquisition tool Acquisition Mode.
Page 328 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Fig. 426 HDR image display (left side without, right with HDR) The Sampling mode is a meant to improve the image display as it equalizes the differences between the very high and the very low intensities in the image.
Page 329 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS After acquisition, the grey dynamics of the image are automatically linearized (see description above) to match the range of the output display. This step visibly intensifies weak signals (Fig. 427).
Page 330 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 During data recording with the Illumination method, masks are applied to protect the sample from laser irradiation. The respective ROIs are displayed in an additional image (Fig. 428).
Page 331: Processing - Stitch
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.13 Processing – Stitch Licensing Instrument standard  optional  LSM  ELYRA Lightsheet Z.1 Tool accepts image data recorded data recorded with the acquisition tool Tile Scan (incl.
Page 332: Processing - Palm
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.14 Processing – PALM Licensing Instrument standard  optional  ELYRA Lightsheet Z.1 Tool exclusively accepts PALM data. See full description of tool set for details.
Page 333: Palm - Convert To Image
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.14.1 PALM – Convert to Image The Convert to Image function allows converting the vector map of centroids to be transformed to a bona fide image.
Page 334: Palm - Import Palm Molecules
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.14.2 PALM – Import PALM Molecules The Import PALM molecules function allows importing PALM molecule tables saved as text files (extension .txt) and reconstruct a PALM image from the table.
Page 335: Palm - Localization Precision
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.14.3 PALM – Localization Precision Localization Precision Select attach Localization Precision look up table (LUT) to an Experimental PSF (Fig. 437). Highlight the Image with the Experimental PSF (should be derived from the PSFs obtained by a 4 µm stack of 100 nm beads) and press Select...
Page 336: Palm - Palm
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.14.4 PALM – PALM The PALM function allows setting filters for peak finding and choosing a fit model for the localization calculations. Select the PALM tab to select the PALM Processing tool (Fig.
Page 337 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS − Account for overlap (multi-emitter): multi- emitter events will be fitted taking into account overlap. selected, Maximum cluster size slider and input box ig. 443 Maximum cluster size field become available (Fig.
Page 338 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The Peak mask size filter defines the radius in pixel units for a detected peak intensity that will be used to cut out the peak and put the peak into a new coordinate system. The size will also be used when fitting a 2D Gauss function to the intensity distribution of the peak.
Page 339 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS If the discard overlapping molecules was selected from the Settings drop down menu, all identified peaks with overlapping circles will be indicated in red and discarded (Fig. 449). If ignore...
Page 340 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The Peak intensity to noise filter defines the threshold of the signal strength of an identified peak over background, at which the peak is regarded to be genuine. The filter works as follows: −...
Page 341 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Expand the Localizer field to have access to the fit model used to localize peaks (Fig. 452). Three fit models are available that can be selected by the Fit Model drop down menu (Fig.
Page 342 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 If the Average before Localization check box remains inactive, every peak (event) will be localized independently and is regarded as being derived from one molecule. Later on, peaks can be...
Page 343 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Expand the Grouping field to have access to the Grouping tool (Fig. 458). You can set two filters to be taken into consideration when peaks (events) are grouped: Fig.
Page 344: Palm - Remove Palm Outliers
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.14.5 PALM – Remove PALM Outliers The Remove PALM outliers function allows removing all molecules that within a defined radius will have less than the specified numbers of neighboring molecules.
Page 345: Processing - Particle Tracking
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.15 Processing – Particle Tracking Licensing Instrument standard  optional  ELYRA Lightsheet Z.1 Tool was exclusively developed for tracking molecules or particles having a dot-like appearance in the image data.
Page 346 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Once the settings are complete press Apply to obtain the trajectories of the single molecules (Fig. 466). Please note, that Particle Tracking only works for 2D data sets.
Page 347: Processing - Experimental Psf
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.16 Processing – Experimental PSF Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts Z-Stacks. See full description of tool for details.
Page 348 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 The PSF sampling pull down menu allows you to interpolate the result to different degrees. Since the resulting PSF is an average of many PSFs...
Page 349 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Feature size sets the mask size that is used to cut out the bead and to create the Experimental PSF. The extensions are displayed in the Preview tab of the image container as circles (Fig. 473).
Page 350 ZEISS Left Tool Area and Hardware Control Tools LSM 880 Once settings have been completed the Preview window displays beads that are not taken into account as red circles and those that are used in white circles (if channel color is grey) or in their channel color (if chanel color is other than grey) (Fig.
Page 351: Processing - Structured Illumination
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.17 Processing – Structured Illumination Licensing Instrument standard  optional  ELYRA Lightsheet Z.1 Tool accepts data recorded in SIM mode on ELYRA. In data sets containing other types of image data as well ("mixed mode"...
Page 352: Structured Illumination - Fourier Transform
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.17.1 Structured Illumination – Fourier Transform The Fourier Transform tool computes and displays the Fourier transform of the image (frequency space). Fig. 476 Structured illumination – Fourier...
Page 353: Structured Illumination - Structured Illumination
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.17.2 Structured Illumination – Structured Illumination The Structured Illumination function allows automatic or manual processing of a set of structured images obtained by phase shift and rotation of the grid.
Page 354 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 The Run Batch button will turn into a Stop Batch button (Fig. 481) once pressed. If the button is pressed, batch processing will be stopped, no matter if Apply was pressed or not and the button turns back to Run Batch.
Page 355 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS If the stack is large, available memory might be limited. In this case a Warning is issued, recommending the usage of the 3D large mode (Fig.
Page 356 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 If the Max.Isotrop checkbox is ticked, the software will compute the high resolution image with the same resolution in every direction that is it does not use the highest frequencies in the rotational directions and the Fourier transform will be circle like (Fig.
Page 357 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Preview tab shows the Input image and a Preview of the output image. By pressing Default the current parameter values will be stored as default values. By pressing Reuse the last stored default values will be loaded.
Page 358: Structured Illumination - Tirf-Filtered
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.17.3 Structured Illumination – TIRF- Filtered This function works for SIM images (stack) in combination with a TIRF image. The lowest frame (first frame) of the stack, preferentially one taken Fig.
Page 359 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The Link drop down menu available for each channel displays all channels of the SIM Input Image (Fig. 500). You can now link the TIRF channel to one of the SIM channels by highlighting the latter.
Page 360: Processing - Channel Alignment
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.18 Processing – Channel Alignment Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts multi-color image data (including multi-dimensional data sets). See full description of tool set for details.
Page 361 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS If the Fit check box is ticked and the Input 2 checkbox inactive, than the software will align all channels of Input image having the same format to the first listed channel of the same format.
Page 362 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Lateral (Fig. 503) is used to correct for linear lateral and axial drifts, that is in all directions. Linear alignments are performed in the x, y and z direction, when the Applied button is pressed.
Page 363 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Using the channel alignment tool for drift corection The following procedure can be used to remove lateral drift in an inage: • Duplicate the time series. Select the time series in the...
Page 364 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Apply drift correction. In the Channel alignment Processing function select the reference time series as input 1 and the original time series as input 2. Check the Fit box and the Input 2 box and leave Marker box unchecked (Fig.
Page 365 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS When the Save Default button is pressed, the present coefficients will be stored and can be recalled by pressing the Restore default button, which becomes active once the Save default button was pressed once.
Page 366 ZEISS Left Tool Area and Hardware Control Tools LSM 880 In either case, the other loaded image does not play a role. In the coefficient table the channel (Ch) assignment column is replaced by an identity (Id) column that numbers the channels in their ascending order starting with 0.
Page 367: Processing - Lightsheet Processing
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.19 Processing – Lightsheet Processing Licensing Instrument standard  optional ELYRA  Lightsheet Z.1 See full description of tool set for details. The Lightsheet Processing module allows one to process images that were acquired with the Lightsheet Z.1 using Dual Side illumination and/or with the Multiview tool tab.
Page 368 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Mean Fusion: The average intensity for each pixel is calculated using the values from both illumination sides. Maximum Fusion: The intensity of each pixel of both illumination images is compared and the value with the higher intensity is used for the resulting image.
Page 369 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Fig. 517 Dual Side Fusion – Fusion Subset in X, Preview and Image control area After pressing Apply, a new .czi file will be generated. The original file name will be extended with "_Dual Side Fusion".
Page 370: Multiview Processing
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.19.2 Multiview Processing 5.4.19.2.1 Deconvolution For the multiview datasets acquired with the Lightsheet Z.1, Deconvolution is an available option (Fig. 518). The following datasets are available for this...
Page 371 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Fast Iterative: The "Fast Iterative" method is an iterative restoration method that uses only one iteration per convolution step (see Meinel, E.S.: Origins of linear and nonlinear recursive restoration algorithms. J.
Page 372 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Adjust Strength checkbox: If you have selected the Nearest Neighbor algorithm or Fast Iterative, the set strength manually checkbox is always activated. With the other Algorithm options, you can activate or deactivate this option using the check box next to Set strength manually.
Page 373 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS To edit any of the input boxes, press the Override button. To get back to the settings provided by the software, press the Master Reset button.
Page 374 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.19.2.2 Registration Options The Registration Options check-box next to Multiview Processing opens the interface to define settings Registration Parameters, the Input settings, and the Fusion options for the data (Fig.
Page 375 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.19.2.2.1 Landmark Alignment For the Landmark Alignment (Fig. 522), the sample must have been embedded with appropriate fluorescent beads (fiducials) which can be recognized by the software. The beads can be present in the same channel as the image information.
Page 376 ZEISS Left Tool Area and Hardware Control Tools LSM 880 For Mean Fusion and Maximum Fusion the checkbox Fusion Subset in X with Preview can be activated (Fig. 523). With these controls it is possible to define which area of the left and the right illumination image should be used for fusion.
Page 377 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Fig. 524 Dual Side Fusion – Fusion Subset in X, Preview and Image control area Ignore Sample: This option activates an additional function to find structures within the sample that were incorrectly labeled as beads (fiducials).
Page 378 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.19.2.2.2 Intensity Based Alignment The Intensity Based Alignment option does not need any fluorescent beads (fiducials) present in the sample since registration is done based on structural information within the image content.
Page 379 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS DFT Fusion (Discrete Fourier Transform Fusion): The Fusion is performed based upon the published method ("Multiple imaging axis microscopy improves resolution for thick-sample applications", Jim Swoger, Jan Huisken, and Ernst H.
Page 380 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Fig. 527 Dual Side Fusion – Fusion Subset in X, Preview and Image control area Expand To Maximum Volume: This checkbox is activated as default. In a multiview experiment not all views might include all the information.
Page 381 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.19.2.2.3 Use Registration From File When the Registration for the dataset has been determined already and the transformation matrix has been saved as an .xml file (Filename.czi.xml), this option can be chosen. Click on the button next to Registration File to open a Windows Explorer window in which you can find and select the file.
Page 382 ZEISS Left Tool Area and Hardware Control Tools LSM 880 To generate the maximum intensity projection image either the Front View or the Side View button can be pressed. In the resulting maximum intensity projection image, each view is handeled as a channel (e.g.
Page 383 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS View 1 is the master view and cannot be moved during the process, the button View 1 is therefore inactive and grayed out. Any of the other views can be activated by pressing the according button (e.g.
Page 384 ZEISS Left Tool Area and Hardware Control Tools LSM 880 Repeat the process with the Right slider and input box; here the marking lines are in blue and the illumination slider in the image control area should be placed to 2, showing the right side illumination of the original data.
Page 385 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Ignore Sample: This option activates an additional function to find structures within the sample that were incorrectly labeled as beads (fiducials). These structures are not taken into account during the Landmark Alignment process;...
Page 386 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.19.2.4 Fusion After Registration of the Views, the Fusion process can be performed (Fig. 534). This will lead to one Z-Stack or Z-Stack over time.
Page 387: Online Multiview Processing
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.19.3 Online Multiview Processing Processing of a multiview experiment dataset can be done online during data acquisition while the data is streamed onto a storage and analysis PC. ZEN software must be started on the storage and analysis PC and the master file (*.czi) of the experiment can be opened.
Page 388: Processing - Copy
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.20 Processing – Copy Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1 Tool accepts all types of image data as input. See full description of tool set for details.
Page 389 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Delete Image Single images or series of images can be deleted from the selected image series (input image) acquired as time series or Z-Stacks or combinations of those.
Page 390: Processing - Ics
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.21 Processing – ICS Licensing Instrument standard  optional  LSM ELYRA Lightsheet Z.1 Tool accepts exclusively input acquired with LSM 710 and LSM 780. See full description of the tool set and section on RICS (Raster Scanning Image Correlation Spectroscopy) for more details on requirements to be met.
Page 391: Remove Structure
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.21.2 Remove Structure • Activate Remove structures in the ICS tool. The Remove structures menu will appear. • To select an image, highlight the image in the image container and press the Select button.
Page 392: Ics Correlation
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.21.3 ICS Correlation • Activate ICS correlation in the ICS tool. The ICS correlation menu will appear (Fig. 542). • Highlight the image in the image container and press the Select button.
Page 393: Map Filter
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.21.4 Map Filter • Activate Map filter in the ICS tool. The Map filter menu will appear (Fig. 545). • To select an image highlight the image in the Image container and press the Select button.
Page 394: Nandb Analysis
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.21.5 NandB Analysis • The function NandB (Number&Brightness) Analysis calculates the image content in two ways: − It counts the photons per second per molecule referred to as the Brightness.
Page 395 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Result Images: Number b, brightness e (molecular); This method calculates the number of − molecules n and their brightness e according to the following formula: ��...
Page 396 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Advanced Options Display Negative Values shifts the offset such that also negative values are displayed as a greylevel above zero. Subset Length (Frames)allows to define subsets of time series and have a value processed for each subset.
Page 397: Processing - Adjust
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.22 Processing – Adjust Licensing Instrument  standard optional  LSM  ELYRA  Lightsheet Z.1* Tool accepts all types of image data as input. See full description of tool set for details and potential restrictions.
Page 398: Interpolate Brightness And Contrast
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.22.2 Interpolate Brightness and Contrast This function permits a continuous contrast and brightness adjustment in a Z-Stack or Z-Stacks over time by interpolation between the starting and end values of the respective image series.
Page 399: Channel Shift
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.4.22.3 Channel Shift Channel shift is used to produce a congruent image with relation to the pixels of the various channels (Fig. 550). This pixel correction function is particularly important in UV applications.
Page 400: Shading Correction
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.4.22.4 Shading Correction Shading Correction is a tool to manipulate images which are unevenly illuminated over the field of view. This works with single images, time series, Z- Stacks, or tiled images.
Page 401: Maintain Tab
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Maintain Tab Press the Maintain tab to have access to the Maintain tool group area (Fig. 552). The maintain tool group allow to adjust hardware settings.
Page 402: Maintain - Adjust Pinhole And Collimators
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.5.1 Maintain – Adjust Pinhole and Collimators Expand the Adjust Pinhole and Collimator tab have access pinhole collimator adjustment (Fig. 553). This function is only available for the LSM imaging mode.
Page 403: Pinholes Section Panel
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.1.1 Pinholes Section Panel The Pinholes Section panel has the following options: Description field Display of the relevant active channel. Diameter [µm] slider Setting of diameter, X-, Y- and Z-position of the pinhole in relation to the beam path (Z-position can be set only for PH1) using the slider or Position X Vis [µm] slider...
Page 404: Collimators Section Panel
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.5.1.2 Collimators Section Panel The Collimators Section panel has the following options (Fig. 555): Name drop down menu Selection of the collimator (IR / VIS or UV / VIS) to be adjusted via the drop down menu.
Page 405: How To Adjust The Pinhole And Collimator
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.1.3 How to Adjust the Pinhole and Collimator Adjustment pinholes performed manually or automatically. Temporary optimization of the adjustment: The position of the pinhole relative to the detector...
Page 406 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 • Click the LSM Continuous action button to start a continuous scan. Optimize the pinhole position in X, Y and Z (Z only for PH1) relative to the PMT using the Position X [µm], Position Y...
Page 407 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS Automatic pinhole adjustment: The automatic adjustment allows the pinholes to be used with any combination of beam splitters and more than one active channel. Please...
Page 408: Maintain - Objectives
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.5.2 Maintain – Objectives Expand the Objective tab to have access to objective definition and selection (Fig. 561). With this function one can assign an objective to its respective position in the nosepiece, e.g.
Page 409: Select Objective
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.2.2 Select Objective Select the Objective tab from the Objective tool, which will show the Name of all objectives with an assigned nosepiece Position. Automatic...
Page 410: Add Objective
Objective directory. Fig. 565 Adding a new Objective – Editing field If the Non Zeiss check box is ticked, objectives from other manufacturers can also be included in the database. If the name of the objective already exists, a warning is displayed telling the user to rename (Fig.
Page 411: Remove Objective
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.2.4 Remove Objective This function allows removing objectives from the User Defined Objective database (Fig. 567). Expand the User Defined Objective directory and highlight the objective you want to delete.
Page 412: Focus Speed
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.5.2.6 Focus Speed Press the Focus Speed tab to have access to the Focus Speed definition window (Fig. 570). The Focus Speed can be changed for every single listed objective.
Page 413: Maintain - Filters
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS For adjustment proceed as follows: Position a thin sample (cell or grid) in the sample holder of the stage and focus with the first objective in the nosepiece.
Page 414: Maintain - Airyscan
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.5.4 Maintain – Airyscan The Airyscan tool provides options for automatic alignment of the Airyscan detector. As a superresolution detector the alignment within the detection beam path is critical and it is...
Page 415: Options - System Options
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.5 Options – System Options The System Options tool allows setting hardware parameters. Expand the tool to have access to the System Options setting window (Fig. 577).
Page 416: System Options - Load Configurations
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.5.5.1 System Options – Load Configurations Press the Load configurations tab to open the Load configurations panel (Fig. 578). Load configuration lists parameters that, when...
Page 417: System Options - Hardware
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.5.3 System Options – Hardware Press the Hardware tab to open the Hardware panel (Fig. 580). Hardware lists parameters that, when their respective check box is ticked, will set several hardware defaults.
Page 418: System Options - Image Display
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 5.5.5.4 System Options – Image Display Press the Image display tab to open the Image display panel (Fig. 581). If the Apply brightness and contrast changes to...
Page 419: Options - Fcs Options
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.6 Options – FCS Options Open the FCS Options tool (Fig. 582). The tool has three tabs: − Data, − Measure, − Auto Save. 5.5.6.1 FCS Options –...
Page 420 ZEISS Left Tool Area and Hardware Control Tools LSM 880 The names of the raw data files are identical to the .fcs measurements, when stored along with them plus an extension that characterizes the measurement. Temporarily stored raw data do not contain the measurement name and have only the extensions.
Page 421 CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS The recorded word has the following structure (Fig. 584): Fig. 584 Structure of the recorded word Two examples should make the format clear (Fig. 585). The tables show part of the running pulse train (from left to right) divided into clock cycles.
Page 422 CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Example 1 - The following words will be recorded: − at W1: high byte: 00011001(bin) = 19 (hex); low byte: 123 (dec) = 7B (hex); resulting word = 7B19 (hex) −...
Page 423: Fcs Options - Measure
CHAPTER 1 - SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 5.5.6.2 FCS Options – Measure Move Stage During Measure If the Move stage during measure check box is activated, the stage will move during a measure- ment (Fig.
Page 424: Options - Service
CHAPTER 1 - SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 Enable Laser Shutter Filter Wheel If additional shutters (before the HeNe lasers and DSSP lasers and a line suppression filter wheel for the Argon laser) are available in the laser module, they can be activated by checking the Enable laser shutter filter wheel check box.
Page 425: Center Screen Area / Image Containers - Display And Image Analysis
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Center Screen Area / Image Containers - Display and Image Analysis Structure and Functional Concept of the Center Screen Area and the Image Display Container 6.1.1...
Page 426 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 591 Image Display window – image tabs show options View tabs: The View tabs make all viewing options and image analysis functions directly available from the main view.
Page 427 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 592 Image Display window; –image tabs show options View Option control tabs: These tabs allow individual activation / deactivation of the available View Option control blocks by clicking on the tabs.
Page 428 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 In order to reset the tab layout – use the entry Reset Tab Layout in the context menu or click on the triangle to access the View Options Area menu.
Page 429: Container Configuration With The Container Context Menu
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Expose Mode: Clicking the Expose Mode button in the top right corner of the Image Display opens a field of thumbnails in the image display with one large thumbnail for each open image.
Page 430: View
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 2D View This function allows to − display a single image in frame mode, − display multiple channel images in superimposed mode.
Page 431 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS The functions of the Zoom and Tools lines are: Zoom Normal: The image is fitted automatically to size of the Image Display window which is always the originally displayed size.
Page 432 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Dimensions – Reuse Clicking the button transfers ALL acquisition parameters (exception: objective and collimator, see below) from the stored image data to the Microscope Hardware Settings / Control tools and applies those parameters directly to the system.
Page 433: Display
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Dimensions – Positions When clicking Positions a crosshair will be displayed at the cursor position in the image. With a mouse click the position of the crosshair in the image will be added to the Positions list in the Stage tool and the Positions tool.
Page 434: Player
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.2.3 Player This function allows to − animate frames of a Z-Stack or a time series − specify animation parameters such as range and animation speed Fig.
Page 435: Graphics
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.2.4 Graphics This function allows to − select from a set of drawing functions such as rectangles and arrows, − add a scale bar to the image, as well as text annotations, −...
Page 436 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 The following list describes the most common functions in the Graphics view control block. More can be found in the pull down menu.
Page 437 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Line thickness pull down: sets the line thickness of the selected element. Color selection pull down: In this pull down, different colors can be assigned to the graphic elements with a click of the mouse.
Page 438: Preview View
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 To insert a Coordinate Label in the image display, click the button in the More … dialog. The coordinate settings dialog (Fig. 608) offers the...
Page 439: Split View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Split View This function allows to − display the individual channels of a multi channel image as well as the superimposed image.
Page 440 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 610 Image Display - Dimensions – Channels with mixed modes In the ELYRA and LSM 7 ELYRA systems channels of different imaging modes can be combined. For example, if SIM (SR channel) or PALM (HR channel) images are computed they can be displayed in combination with widefield (WF channel) or sum widefield (SWF channel) images (Fig.
Page 441 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS If group channels is selected all channels belonging to the same color will be merged in one image (Fig. 613). Fig. 613 Image Display, Split view with mixed modes –...
Page 442: Gallery View
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Gallery View This function allows to: − display images (Z-Stack, time series, combination of both) side by side in a tiled fashion, −...
Page 443: Airyscan View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Airyscan View When data are acquired with Airyscan, the Airyscan View Tab becomes available. 6.5.1 Airyscan – View Option Tab Dimensions All Airyscan display and online processing functions can be done in the Airyscan tab (Fig.
Page 444: Airyscan - View Option Tab Display
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.5.2 Airyscan – View Option Tab Display Raw data and SR preview Due to the nature of the Airyscan detection and the signal reassignment to the respective airy orders, the display curves (histogram) of the source image and the processing preview are very different.
Page 445: Airyscan - View Option Tab Airyscan
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.5.3 Airyscan – View Option Tab Airyscan 2D Processing Image preview There are several options available for the display of the Airyscan data.
Page 446 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Detector view Shows the intensity distribution of the detection elements. This also allows a judgment whether the system is adjusted correctly (see section Maintain Tab). The brightest element should be in the center with a drop of the signal towards the periphery.
Page 447 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Slightly reducing the SR filter strength will result in an optimal result (Fig. 625). Typically, this position is 0.3 to 0.5 units higher than the Auto default value.
Page 448 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Parameters O(r): object, D(r): image, N(r): noise, EH(r): PSFeff, O(ω): object spectrum, D(ω): image spectrum, N(ω): noise spectrum, EH(ω): OTFeff, i: index of detector elements, n: number of detector elements, w =...
Page 449: Ortho View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Ortho View This function allows to − display a Z-Stack of images in an orthogonal view − measure distances in three dimensions The settings of the Dimensions, Display, Player and Graphics view options control blocks apply.
Page 450: Ortho - Select Function
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.6.1 Ortho – Select Function • By changing the parameters X, Y and Z in the Ortho View Option control block, the section plane can be positioned at any XYZ coordinate of the Z-Stack.
Page 451: Ortho - Distance Function
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.6.2 Ortho – Distance Function • Activating the tick box permits length measurements in 3D space. • Click on the button to set the first XYZ-point for the measurement of the spatial distance.
Page 452: Cut View
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Cut View This function allows to display a user defined section plane (= cut plane) of a Z-Stack. It automatically improves the image of the section plane by trilinear interpolation.
Page 453: D View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 2.5 D View This function allows to − display the two-dimensional intensity distribution of an image in an pseudo 3D mode, −...
Page 454 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 633 2.5 D Image Display The 2.5 D view control tab contains the following function elements: Profile button Profile display (vertical polygon display). Setting of the Profile Distance between 1 and 20 using the slider.
Page 455: View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 3D View Use this View tab to display image Z-Stacks in three dimensions. Display and rendering of Z-Stacks with up to eight color channels are supported and the data can be a time lapse series (5D Image Stack).
Page 456 ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 On the 3D View control panel the different render modes are accessible: Shadow, Transparent, Maximum, Surface and Mixed. The rendering quality can be adjusted by using slider.
Page 457: Shadow Projection
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Series: 3D Data can be visualized online by simply grabbing and turning the data stack with the mouse. In addition to this interactive way of interacting with the data 3D VisArt is able to perform render series where the dataset is turned around one of the axis, along a freely defined position list or a freely definable start and end point.
Page 458 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 637 Image Display window, 3D display, Shadow projection, Front view The zoom wheel to the left of the Image Display window allows continuous zooming of the 3D reconstructed image.
Page 459 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS The following additional buttons are available in the Any View shadow projection mode: • After activation of the Frame button (below the image), a bounding box is drawn around the 3D reconstructed image.
Page 460: Transparency Render Mode
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.9.2 Transparency Render Mode Principle: In the Transparent mode a three-dimensional image with a transparent effect is calculated. At least two-dimensional...
Page 461: Maximum Mode
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS By means of the Depth Coding function (Fig. 641), the Z-information contained in a sequence can be colored with the colors of the rainbow, in which case "blue"...
Page 462: Mixed Render Mode
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Basic button Surface rendering (voxel based) Advanced button Surface rendering (triangle based) Full Resolution button High accuracy surface rendering (triangle based) 6.9.5...
Page 463: Clipping Planes
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.9.6 Clipping Planes Fig. 645 Image Display window, 3D display, Clipping planes 3D VisArt can display up to three so called Clipping planes. These planes can be used to cut the volume open in order to visualize structures which are within a given volume.
Page 464 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 All these functions can be used for creating a render series in order to show them and to make spectacular movies for presentations.
Page 465 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS These adjustments can be made individually for each plane. The clipping planes can be chosen by selecting the respective button. Appearance of the clipping planes By default the clipping planes have an opaque surface and a colored outline.
Page 466 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Clipping Planes Settings Activate or deactivate the clipping functionality globally by the check box Enable clipping. displays the three clipping planes in the default mode (opaque surface, colored outlines, clipping planes active, no clipping).
Page 467: Flying Mode
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.9.7 Flying Mode The Flying mode is an interactive way to explore three-dimensional data. By pressing the mouse wheel you can fly into and through a dataset. Moving the mouse left / right and up / down controls the flying direction.
Page 468 ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 − Roughness (for Shadow mode only): You can also influence the roughness used to display the surface of the image structures. This changes the plasticity of the display (relative scale between 0 and 1).
Page 469: Series
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.9.9 Series Fig. 654 Series tab The Series tab (Fig. 654) displays the Render Series control panel. This panel allows settings for the axis to be used for rotation of the 3D reconstructed images.
Page 470 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Position List mode In the Position List mode, the image is reconstructed between any required number of interim positions to be determined individually. Just...
Page 471: Interactive Measurements
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.9.10 Interactive Measurements 3D VisArt features three tools for interactive measurements in Euclidian space. Measurements can be performed in all graphic-card accelerated modes (Transparency, Surface, Maximum and Mixed). The measured tools are shown as shapes in the rendered volume and can be changed there interactively.
Page 472: Settings
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.9.11 Settings Flying mode Ticking the tick box Flying mode activates the mode, unticking deactivates it. To show or hide an overview image tick the Show overlay image box.
Page 473: Options
Renderer: OpenGL Software is rendering the 3D data with the CPU; OpenGL Hardware uses the Graphics card to do this. Especially when having trouble with non Zeiss-certified graphics cards it is sometimes useful to switch to a pure CPU based rendering.
Page 474: View - Basic
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Intensity Alpha textures: This feature optimizes the usage of the graphic ram with single channel images. Display Lists activates a batch processing of the operations the graphics card is doing. It results in faster rendering.
Page 475: Histogram View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.10 Histogram View The Histogram View (Histo View) allows to − display a histogram (distribution of pixel intensities) of an image or Region of Interest, −...
Page 476 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 666 Image Display, Histogram view The Histo button can also be used online during scanning. 000000-2071-464 10/2014 V_01...
Page 477 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Histogram functions: Skip Black and Skip White tick boxes: Ignore black pixels (gray value 0) and Ignore white pixels (gray value 255 or 4096) in the histogram.
Page 478: Colocalization View
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Histogram Threshold: When the Threshold tick box is selected, the controls to set a lower and upper threshold for the histogram calculation are displayed (Fig. 667). The threshold can be set for each channel (channel selection pull down menu).
Page 479 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 669 Image Display, Colocalization view Fig. 670 Scatter diagram and threshold with crosshair 10/2014 V_01 000000-2071-464...
Page 480: How A Scatter Diagram Is Generated
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.11.1 How a Scatter Diagram is Generated All pixels having the same positions in both images are considered a pair. Of every pair of pixels (P1, P2) from the two source images, the intensity level of pixel P1 is interpreted as X coordinate, and that of pixel P2 as Y coordinate of the scatter diagram.
Page 481: Quantitative Colocalization Parameters Shown In The Data Table
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.11.2 Quantitative Colocalization Parameters Shown in the Data Table No. of pixels in image ROI or scatter region − Area / relative area of image ROI or scatter region −...
Page 482: Overlap Coefficient, Overlap Coefficient After Manders
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.11.4 Overlap Coefficient, Overlap Coefficient after Manders (Manders, Verbeek and Aten, J. Microscopy 169:375-382, 1993) ∑ ∑ ∑ Another parameter used to quantify colocalization in image pairs −...
Page 483 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 672 Image Display - Profile View, Line Profile with markers Fig. 673 Image Display - Profile View, Profile displayed in Image...
Page 484 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Control block tools The Profile toolbar contains the following tools: Select button: Activates the mouse button for selection, resizing or movement of the profile line in the Image Display window.
Page 485: Shuttle And Find View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.13 Shuttle and Find View Within the S&F control tab of the Shuttle and Find view (Fig. 674) regions of interests as well as points of interest can be defined.
Page 486 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Regions and Find functions Button Description Select By clicking on this button you activate the selection mode (button is marked blue) to select the ROIs or POIs in the image area (if drawn in).
Page 487 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Graphical elements list List Columns Description Eye symbol Shows or hides the ROI / POI in the image. Lock symbol Locks a ROI / POI to prevent changes.
Page 488: Mean Of Roi: Additional View Type For Time Series
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.14 Mean of ROI: Additional View Type for Time Series The Mean of ROI View Type allows to − display the intensity - time diagram (mean intensity in user defined ROIs over time), −...
Page 489 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 676 Image Display window, Mean ROI display for time series in single plane Fig. 677 Image Display window, Mean ROI display for time series of Z-Stack...
Page 490 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 The Mean of ROI view options control block contains the following function elements: ROIs can be managed in the Graphics View Option control block.
Page 491 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Buttons for diagram display options: Mode pull down: choose between area and mean mode Area: Display of the area of the ROI in the intensity time diagram, depending on the set threshold values.
Page 492: Kinetic / Frap View: Additional View Type For Time Series
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.15 Kinetic / FRAP View: Additional View Type for Time Series The FRAP function permits interactive analysis of bleaching experiments, including −...
Page 493 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Available tools in the FRAP View: Channels Select single channels or all channels for analysis in the general Dimensions view options control box.
Page 494 ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Example: FRAP Performed in a Nucleus Expressing GFP Labeled Proteins Display of the image series in the Mean ROI display mode: The drawing tools are used to define the ROI to be analyzed (ROI 1), the background ROI (ROI 3), and the reference ROI (ROI 2) (see Fig.
Page 495 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS • Switch to the FRAP View tab. • Mark the check boxes for background and reference region. • The intensity values of the ROI to be analyzed will be corrected for background intensity and changes in intensity calculated for the reference region.
Page 496 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 681 Image window displaying the analysis of FRAP data using a mono exponential fit Fig. 682 Image window displaying the analysis of FRAP data using a...
Page 497 A more general introduction to FRAP experiments can be found in the "Methods" brochure "Photomanipulation with Laser Scanning Microscopes" by Carl Zeiss Microscopy GmbH (order no. 45- 0060 e) and the references cited therein.
Page 498: Lambda Coded: Additional View Types For Lambda Mode
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.16 Lambda Coded: Additional View Types for Lambda Mode The Lambda Coded View displays a Lambda Stack in a wavelength-coded color view. A color palette is automatically assigned to the individual images which are then displayed in a merge-type display.
Page 499 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 685 Dimensions view option control block for Lambda Coded View 10/2014 V_01 000000-2071-464...
Page 500: Unmixing View
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.17 Unmixing View In the Unmixing View, Lambda or channel data sets can be analyzed and spectrally unmixed (for more information see as well section Processing –...
Page 501 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 688 Unmixing Display of a Lambda stack acquired on an LSM 710 / LSM 780 10/2014 V_01 000000-2071-464...
Page 502 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 689 Unmixing Display of a Lambda stack with a spectral dataset acquired on an LSM 700 with a variable secondary dichroic...
Page 503 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Tools in the Unmixing View Options Control Block − Tools for display customization and drawing: Drawing tools work the same as in the Graphics View Option control...
Page 504 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 An additional channel per unmixed component is displayed which shows the relative standard deviation (RSD) in each unmixed component channel (see also section Processing – Linear Unmixing).
Page 505: Automatic Component Extraction
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.17.1 Automatic Component Extraction The use of this function permits the automatic search for the individual reference spectra in a Lambda stack.
Page 506 ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Tools in the Unmixing - Extract View Options Control Block: These tools (Unmixing – Extract) are only available for Lambda stacks on an LSM 710 and LSM 780 system with 34 channel Quasar detector.
Page 507: Fret View
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.18 FRET View In the FRET View type (Fig. 693), FRET data sets can be analyzed: − Data gained performing Acceptor Photobleaching −...
Page 508 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 FRETN (p): Result of analysis using Gordon method. The FRETN value is calculated for each pixel, afterwards the average for the chosen region is determined.
Page 509: Tools In The Fret View Options Control Block For Acceptor Photobleaching
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.18.1 Tools in the FRET View Options Control Block Acceptor Photobleaching FRET tab (Fig. 695): Method Acceptor FRET Analysis, Photobleach is automatically selected in the drop down list.
Page 510 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Thresholds tab (Fig. 697): The threshold for the image analysis can be set manually using the slider or editing text box next to Donor, Acceptor or All, where All moves both sliders to the same value.
Page 511: Tools In The Fret View Options Control Block For Sensitized Emission
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.18.2 Tools in the FRET View Options Control Block Sensitized Emission FRET tab (Fig. 700): Within this tab overlay regions can be defined.
Page 512 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 N-FRET or Xia method: Xia and Liu, Biophys J 81, 2395 (2001) NFRET = FRET 1/ √(Dfd*Afa) Displays an N-FRET image with intensities converted from the FRET index calculated for each pixel using the Xia method.
Page 513 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Thresholds tab (Fig. 702): The threshold for the image analysis can be set manually using the slider or editing text box next to Donor, Acceptor, FRET or All, where All moves all sliders to the same value.
Page 514: Raster Scanning Image Correlation Spectroscopy (Rics)
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.19 Raster Scanning Image Correlation Spectroscopy (RICS) RICS is an image analysis tool that allows retrieving dynamic information from intensity images or image stacks.
Page 515: Access To Rics
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.19.1 Access to RICS If you have taken an image or an image series or have opened an old image or image stack, a click on the RICS tab of the View tab list will make the RICS functionality available.
Page 516: Correlation Functionalities
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.19.3 Correlation Functionalities Activate Correlation under the RICS view. The Correlation options will be displayed in the View control window (Fig. 705).
Page 517 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 706 Image window with two selected regions of interest in the intensity image Correlation allows you to select the type of correlation analysis you want to perform.
Page 518 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 For a two channel cross-correlation setup, two drop-down menus will be displayed (Fig. 709). The upper channel will be the one that will be cross- correlated against the lower channel.
Page 519: Display Functionalities
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS When Immobile structures (+Stack) is selected, the average image from the stack (pixel by pixel) is subtracted from every image and a scalar is added to avoid negative pixels. The scalar is computed from the average of all pixels from all frames of the stack.
Page 520 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Fig. 716 Correlation displayed clockwise as Diagram, Image, Table and 2.5 D Note, that for different representations different options will become available.
Page 521 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS For the Diagram and Table, the X and Y pull down menu and the Draw symmetric curve check box will be additionally available. In the X and Y pull...
Page 522 ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Checking and unchecking the Show fit-results box will toggle between the correlation and fit images. Note, if this box is activated, the Interpolate G(0) box will disappear. Pressing the export button below the Show fit-results box will export the correlation image or fit image, whatever is active as an image in .lsm format.
Page 523 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS If you press the Settings button, the settings menu will pop up (Fig. 723). In Map Selection you can determine the map type with the Map type pull down menu (Fig.
Page 524 ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 In the Show all mode you have further the option to display the Std.Dev. (standard deviation) maps of both parameter maps as well as the Χ...
Page 525 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS The map size in terms of region numbers is displayed in the info area of the Settings menu. By pressing Generate a map is created with region and shift sizes as determined in the settings.
Page 526: Fitting Functionalities
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.19.5 Fitting Functionalities Activate Fitting under the RICS view. The Fit options will be displayed in the View control window (Fig. 727).
Page 527 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS You can select a model from the Model pull down menu in the Fit table. Every stored model will be listed (Fig. 729). Below the Model pull down menu the active channel is displayed in the Channel display area.
Page 528 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 By clicking with the right mouse button into the Result table the table’s menu box will pop up (Fig. 733). You have the option to copy the table to the clipboard by selecting Copy, or by choosing save to save the data as a text file.
Page 529: Model Functionalities
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.19.6 Model Functionalities Activate Model under the RICS view. The Model options will be displayed in the View control window (Fig. 736).
Page 530 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 For three of the terms (G , A and G ) settings menus are available. To open a settings menu, press the corresponding Settings button.
Page 531 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Translation G (ξ,ψ) describes the translational diffusion term of the model function. To open the Translation settings press the Settings button (Fig. 742).
Page 532 CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 Components allow selecting 1, 2 or 3 components by pressing the respective button. Free / anomalous allow via pull- down menus to select between free Fig.
Page 533: Polarization Imaging
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.20 Polarization Imaging Linear (plane) polarized light, which is light whose wave goes only one direction, exciting a fluorescent molecule with a preferred dipole orientation results in polarized emitted light. It provides a contrast- enhancing method that is especially useful in the study of molecules that are fixed in their orientation or are greatly restricted in their rotational diffusion.
Page 534: Imaging Setup For Polarization Imaging
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.20.1 Imaging Setup Polarization Imaging In case emission polarizers (analyzers) are available, you have a Notch filter cascade selection button available situated on top of the MBS button (see Fig.
Page 535: Determination Of G-Factor
CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS 6.20.2 Determination of G-factor In order to determine the G-factor for the LSM 710 and LSM 780 systems, you need an isotropic medium like a fluorescent dye at a 1 mM concentration, for example fluorescein.
Page 536: Obtaining An Anisotropy Image
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.20.3 Obtaining an Anisotropy Image Set up a multitrack configuration with alternating analyzers: P in track 1, S in track 2, leaving all other settings equal (see Fig.
Page 537 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS In the Ratio1 channel choose the Ratio Type 3 formula. With Ch1-T1 and Ch1-T2 corresponding to I and I this formula corresponds to the formula for anisotropy r. For the factors put in the value for the G- factor in the numerator and 2 x G in the denominator.
Page 538: Information View
CHAPTER 1 - SYSTEM OPERATION ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880 6.21 Information View The Information View is a view type that shows a summary information sheet of all relevant image acquisition parameters. Parts of it can be edited – like, e.g., the Notes field.
Page 539 CHAPTER 1 - SYSTEM OPERATION LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS Fig. 751 Information View of a processed superresolution image 10/2014 V_01 000000-2071-464...
Page 540: Right Tool Area, Data Management And Storage
When exporting .czi files as .lsm files, each z-stack will be saved as an individual .lsm file. We provide further information about the .czi file format on request. Please contact your ZEISS representative. In ZEN there are three ways to access your data: The File menu −...
Page 541: Zen File Browser
CHAPTER 1 - SYSTEM OPERATION LSM 880 Right Tool Area, Data Management and Storage ZEISS ZEN File Browser The ZEN File Browser is a versatile tool to access your image data. • In the menu bar, click on File and select the File Browser … item to open the ZEN File Browser.
Page 542 CHAPTER 1 - SYSTEM OPERATION ZEISS Right Tool Area, Data Management and Storage LSM 880 Eventual info messages (Fig. 753) fading in the right hand corner inform about missing hardware if applicable. The message box disappears after ~ 10 seconds.
Page 543 CHAPTER 1 - SYSTEM OPERATION LSM 880 Right Tool Area, Data Management and Storage ZEISS Within the dialog (Fig. 756) use the editing boxes or the sliders to set the desired values. slider toggles with highlighted dimension. Click Select file name and save… to save the cropped images as separate .czi or .lsm files.
Page 544: Gallery View Of The Zen File Browser
CHAPTER 1 - SYSTEM OPERATION ZEISS Right Tool Area, Data Management and Storage LSM 880 7.2.1 Gallery View of the ZEN File Browser In the Gallery View of the browser the images are displayed as thumbnails. One click selects a single image, press Shift and click to select multiple images.
Page 545 CHAPTER 1 - SYSTEM OPERATION LSM 880 Right Tool Area, Data Management and Storage ZEISS Fig. 759 Images with information 10/2014 V_01 000000-2071-464...
Page 546: Form View Of The Zen File Browser
CHAPTER 1 - SYSTEM OPERATION ZEISS Right Tool Area, Data Management and Storage LSM 880 7.2.2 Form View of the ZEN File Browser All images in a given folder can be accessed by using the slider below the Info-Panel or using the stepper on the right side of the slider.
Page 547: Table View Of The Zen File Browser
CHAPTER 1 - SYSTEM OPERATION LSM 880 Right Tool Area, Data Management and Storage ZEISS 7.2.3 Table View of the ZEN File Browser The Table View is especially useful in case a lot of files are found in one folder.
Page 548: Images And Documents Panel
CHAPTER 1 - SYSTEM OPERATION ZEISS Right Tool Area, Data Management and Storage LSM 880 Images and Documents Panel The Images and Documents panel (Fig. 763) is located in the Right Tool Area of ZEN. It displays all images and data which are open in ZEN. Its purpose is multi-fold: −...
Page 549 CHAPTER 1 - SYSTEM OPERATION LSM 880 Right Tool Area, Data Management and Storage ZEISS The context menu of the Images and Documents section allows the following functions: Selecting one of the view options Textual − View, Thumbnail View or Big View Selecting all opened image files by clicking −...
Page 550 CHAPTER 1 - SYSTEM OPERATION ZEISS Right Tool Area, Data Management and Storage LSM 880 − A progress bar (Fig. 767) is shown for each image facilitating the overview if e.g. many processing function are running at the same time.
Page 551: Opening Of Files Via The "Open" Command In The File Menu
CHAPTER 1 - SYSTEM OPERATION LSM 880 Right Tool Area, Data Management and Storage ZEISS Opening of Files via the "Open" Command in the File Menu • Click on the File menu of the menu bar and select Open or click on the icon of the main toolbar.
Page 552: Export Of Images
CHAPTER 1 - SYSTEM OPERATION ZEISS Right Tool Area, Data Management and Storage LSM 880 Export of Images The Export function allows the export of acquired images and images loaded. • Select the image to be exported. • Click on the Export menu Item in the File menu of the menu bar.
Page 553: Index
CHAPTER 1 - SYSTEM OPERATION LSM 880 Index ZEISS Index Arrow down button ........47 Auto save ............ 418 2D View ............424 Automatic Component Extraction ....499 Automatic dust filter ........272 Average ........108, 110, 304 Averaging selection ........103 32 channel module light path ......
Page 554 CHAPTER 1 - SYSTEM OPERATION ZEISS Index LSM 880 Subset ..........382, 383 Filter ............305 Copy channel ..........382 Filter cube selection ........86 Correction ........... 148 Filters ............407 Correlation ........195, 224, 319 Find focus ............73 Correlation function ........196 First/Last mode ..........
Page 555 CHAPTER 1 - SYSTEM OPERATION LSM 880 Index ZEISS Image tabs ............ 46 Look Up Table (LUT) ........117 Image view controls ........46 Lowering objective ........166 Image view tabs ..........46 Images and documents panel ....... 542 Imaging configuration ........42 Main tool tabs ..........
Page 556 CHAPTER 1 - SYSTEM OPERATION ZEISS Index LSM 880 Sample carrier ......... 229 HDR-imaging ........... 321 Stage ............. 228 ICS ............384 NDD light path ..........181 Image calculator ........302 Nearest neighbour ........364 Ion concentration ........313 New image document button ......48 Lightsheet processing ......
Page 557 CHAPTER 1 - SYSTEM OPERATION LSM 880 Index ZEISS Acquisition ..........190 Time series .......... 155, 236 Correlator ..........224 TIRF-Filtered ..........352 Fit ............192 Titration calibration ........318 Imaging Setup........... 84 Tool .............. 43 Laser ............177 Tool groups ........... 41 Light path ..........
Page 558 CHAPTER 1 - SYSTEM OPERATION ZEISS Index LSM 880 Zoom function ........275, 284 Z-Stack ............141 Z-Piezo ............146 Z-Stack settings for Airyscan ......149 000000-2071-464 10/2014 V_01...
Page 559 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Contents ZEISS CHAPTER 2 MACROS AND VISUAL BASIC CONTENTS Page VBA Version ......................3 Macros ........................3 Macro Language ........................ 3 Macro Control ........................4 2.2.1 Edit Macro Function ......................4 2.2.2...
Page 560 CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Contents LSM 880 Example Macros ........................ 33 4.8.1 Access to images ....................... 33 4.8.2 Backup recording settings ....................33 4.8.3 Event handling ........................33 4.8.4 Export, Save and Load ....................... 33 4.8.5 VBA-GUI API ........................
Page 561: Vba Version
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 VBA Version ZEISS VBA Version Both versions of VBA (Visual Basic for Applications) – version 6.4 (32 bit) and version 7.1 (64 bit) – are available. Version 6.4 is registered by default to ensure compatibility to existing macros.
Page 562: Macro Control
Macros LSM 880 The VBA functionality described in this paragraph refers to VBA as implemented in the ZEISS LSM software 4.2. With the introduction of ZEN, the range of functions is and will be extended, while VBA can still be used with the functions described below. The extension of functionality applies mostly to display and access of image data.
Page 563 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Macros ZEISS Macros are stored and managed in project files (*.lvb). Before you can record or edit a macro, you have to create a project as follows: • Press the New button to create a project file (see Fig.
Page 564 CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Macros LSM 880 Recording panel Before recording a command sequence, you can enter the name for the macro to be created in the Name input box of the Recording panel. Start button: Starts recording.
Page 565: Assign Macro To The Macro List
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Macros ZEISS Proceed as follows to edit a macro: • Select the required macro from the Project list box of the Macros panel. • Click on the Edit button. The Microsoft Visual Basic editing window will be opened.
Page 566: Editing And Debugging Of Macros
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Macros LSM 880 2.2.3 Editing and Debugging of Macros The Edit in the Edit Macro panel button activates IDE (Integrated Development Environment), which allows macros to be edited and debugged. Under the Help - Macrosoft Visual Basics Help menu item, IDE contains detailed "online"...
Page 567: Overview Of Available Macros
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Macros ZEISS Overview of Available Macros Documentation files (*.rtf, *.doc) of advanced macros are located in the macro directory. Additional Macros for Service are listed in a separate folder. Name Description CameraColor60.lvb...
Page 568: Sample Macros
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Macros LSM 880 Name Description SphericObjectiveCorrection60.lvb Calibrates the spheric component of objective error by automatic acquisition of a Z-Stack on a flat surface and a spherical fit of the topographic data. TestGrid60.lvb Projects a testgrid into the image window (password protected).
Page 569: Excitationfingerprinting
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Macros ZEISS ExcitationFingerprinting With tunable multiphoton lasers (except OPO devices) it is possible to acquire Excitation Spectra and subsequently use these spectra to perform linear unmixing. This process is called Excitation Fingerprinting.
Page 570 CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Macros LSM 880 Fig. 9 Macro: Excitation Fingerprinting 1) Chose the AOM Calibration tab. 2) Set AOM Transmission to a value around 10. 3) Set the laser to the lowest wavelength possible (ideally this has already been done in the main software).
Page 571 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Macros ZEISS The normalized AOM values will provide the right AOM settings starting with the chosen setting at a given wavelength and calculating the relative values for all other wavelengths. Thus it is avoided to reach maximum or minimum AOM settings before the end of the wavelength range.
Page 572: Concatenateimages
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Macros LSM 880 ConcatenateImages The mcacro allows concatenating various types of images into one image document showing the final image as a time series image. It is useful to combine images from Multi Block experiments into one image document showing the time series of one Block only.
Page 573: Visual Macro Editor
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Visual Macro Editor ZEISS Visual Macro Editor The Visual Macro Editor is an optional software module which allows the user to program a variety of scanning procedures, image calculations or image processing routines as well as the combination of these functions.
Page 574: Function Description
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Visual Macro Editor LSM 880 Function Description Toolbar buttons Starts the macro starting with the first block in the program flow column. Stop Stops the macro at any time. Break Interrupts the macro. Clicking the button again will resume the actual program flow of the Macro.
Page 575 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Visual Macro Editor ZEISS • Click at the action block with the left mouse button (hold down the mouse button) and drag the mouse cursor into the flow column. Release the mouse button.
Page 576 CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Visual Macro Editor LSM 880 Acquisition Action Blocks Loads a Beam Path Configuration form the Configuration list or uses the current one that is set up in the main program. Use the pull down menu in the Property/Value list to select the Configuration that should be used for imaging.
Page 577 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Visual Macro Editor ZEISS This block performs the Find function according to the main software. This action block sets the Z-position according to the focusing procedure set with the parameters available. The image generated displays the image or image series aquired to set the focus position.
Page 578 CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Visual Macro Editor LSM 880 It is possible to export images choosing a certain file format from the pull down list that appears when highlighting the line File Format in the properties list.
Page 579 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Visual Macro Editor ZEISS Process Action Blocks This block allows to do image calculation using the pixel intensities of the image. The operators for the formulas that can be used are described in the Ratio Dialogue within the Process Menu.
Page 580 CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Visual Macro Editor LSM 880 This action block defines a mask (image region with defined properties) derived from an image window. The image can be a newly acquired or imported image. The properties of the mask based on the image data can be selected according to the following properties: −...
Page 581: Editing A Macro
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Visual Macro Editor ZEISS Sub Macro Actions Blocks This block allows setting a sub macro within a main macro. The sup macro is opened by clicking onto the arrow in the block icon and closed clicking the top right of the program flow column.
Page 582 CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS Visual Macro Editor LSM 880 • Assign the value 1 to a variable named Index (see Fig. 13). Fig. 13 Assignment of the variable • Click the single action blocks and assign the...
Page 583 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 Visual Macro Editor ZEISS • The movement of the stage is defined highlighting the Move stage action block, chosing the Mode Relative Position in the Property list and typing in the value 10 for X...
Page 584: Vba Programming For Lsm
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS VBA Programming for LSM LSM 880 VBA Programming for LSM VBA Programming Visual Basic for Applications (VBA) is a widely-spread programming language, and lots of sources for an introduction to the language can be found on the web. Therefore, we do not give a general introduction into VBA programming here.
Page 585: Object Browser
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 VBA Programming for LSM ZEISS Object browser To search the structure of an API object, use the object browser from the VBA editor. To explore an object from your VBA code, position the curser in the object and press "F2".
Page 586: Glossary
ZEN system. There are two COM-automation objects available, for the Lsm5Vba interface and for the VBA-GUI interface. The COM object Lsm5Vba is called "Lsm5Vba.Application", and the one for the VBA-GUI interface is called "Zeiss.Micro.AIM.ApplicationInterface". For example to connect a MATLAB session to ZEN, start ZEN first, start MATLAB, and then type >>...
Page 587 CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 VBA Programming for LSM ZEISS Application Lsm5Application Lsm5 Lsm5VbaProject CpAmplifiers Lsm5Info CpCollimators Lsm5Tools CpFilterSets Lsm5Constants CpFocus CpIntegrators Lsm5Options CpLamps Lsm5Hardware CpLaserLines CpLasers DsGuidedModeDatabase CpObjectiveRevolvers DsFluorescenseDatabase CpPinholes DsMruList CpPmts DsRecordingDoc CpScanControl CpServos...
Page 588: Programming With The Vba-Gui Interface
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS VBA Programming for LSM LSM 880 Programming with the VBA-GUI interface 4.7.1 Introduction The ZEN VBA-GUI interface allows you to program ZEN with the same comfort and ease as using the mouse to control the GUI. All capabilities of the ZEN system are accessible via an intuitive syntax.
Page 589: Structure Of A Vba-Gui Command
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 VBA Programming for LSM ZEISS 4.7.2 Structure of a VBA-GUI command There are three different kinds of actions that can be performed in the ZEN GUI: Setting a value (either a number or a string, e.g.
Page 590: Selecting And Managing Image Documents
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS VBA Programming for LSM LSM 880 Fig. 23 ZEN VBA-GUI Interface - Editor 4.7.4 Selecting and managing image documents Image document handling in the VBA-GUI interface happens via the "GUI.Document" command. From the list of open documents, images can be selected by index and by name, they can be closed and their acquisition parameters can be re-used.
Page 591: Example Macros
CHAPTER 2 - MACROS AND VISUAL BASIC LSM 880 VBA Programming for LSM ZEISS Example Macros This library of example macros is designed as a starting point for your own programming ventures. They are deliberately kept simple for quick understanding and hand-on. We recommend that you create copies the macros and then edit them to see how ZEN reacts to your commands.
Page 592: Vba-Gui Api
CHAPTER 2 - MACROS AND VISUAL BASIC ZEISS VBA Programming for LSM LSM 880 4.8.5 VBA-GUI API We have not created example macros for the VBA-GUI API, because these can easily be generated by the macro recorder functionality. Activate macro recording and the execute the actions that you want to automate.
Page 593 CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS LSM 880 Contents ZEISS CHAPTER 3 ADDITIONAL SOFTWARE TOOLS CONTENTS Page Stand Select ......................2 SystemMaintenance Tool ..................3 Calibration Objective ......................3 Starting the Maintenance Tool .................... 4 General Operation of the Maintenance Tool ................ 5 ZEN Configuration Tool ..................
Page 594: Stand Select
CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS ZEISS Stand Select LSM 880 Stand Select The Stand Select tool permits a new or updated database to be assigned to the ZEN software program. Fig. 1 Select Stand Database window • Close the ZEN software program and double- click on the Stand Select icon on the desktop.
Page 595: Systemmaintenance Tool
CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS LSM 880 SystemMaintenance Tool ZEISS SystemMaintenance Tool With a calibration objective and correct system configuration, the SystemMaintenance tool allows convenient self-adjustment of LSM. The optical beampath, relative pinhole position and scanner adjustment can be set and checked automatically with this tool.
Page 596: Starting The Maintenance Tool
CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS ZEISS SystemMaintenance Tool LSM 880 Starting the Maintenance Tool Before starting the Maintenance Tool, the system hardware must be fully activated. To do this, open ZEN and check the following settings: Correct entry of all existing lenses; the calibration objective needs to be entered as APO Calibration LSM.
Page 597: General Operation Of The Maintenance Tool
CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS LSM 880 SystemMaintenance Tool ZEISS The Maintenance Tool can only be used, if the calibration objective is entered as APO Calibration LSM. If this is not the case, a message will remind the user to select the calibration objective.
Page 598 CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS ZEISS SystemMaintenance Tool LSM 880 Fig. 9 Maintenance Tool main window Fig. 10 The Maintenance Tool guides users through required adjustment steps 000000-2071-464 10/2014 V_01...
Page 599: Zen Configuration Tool
CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS LSM 880 ZEN Configuration Tool ZEISS ZEN Configuration Tool The configuration tool provides access to the database of a system. Be aware that wrong entries can lead to a malfunction of the system. Always keep a backup of the original database. The database of the system is stored in C:/ZEN/database.
Page 600 ZEISS ZEN Configuration Tool LSM 880 • Within the appearing window (Fig. 12) choose the tabs Scan Head, LSM 880, Filter. Fig. 12 Configuration Tool with tabs seleceted for access to filter settings • Do not change any existing filters or filters from filterwheels inside the scan head. This leads to malfunctions of the system! •...
Page 601 CHAPTER 3 - ADDITIONAL SOFTWARE TOOLS LSM 880 ZEN Configuration Tool ZEISS After selecting the "Filterwheel", the current filters are indicated each with a button next to it referring to the detection channel it is effective for (Fig. 12). • Clicking this button opens the Change Filter window with a list of favourite, potential and user defined filters (Fig.
Page 602 Fig. 15 Add new emission filter window • Indicate the new filter as Non Zeiss and fill in the form to provide all necessary information to the system so it can use the information for image acquisition and processing. The form on how to fill in the information is displayed as example in the lower part of the window when editing a special entry.
Page 603 CHAPTER 4 - ANNEX LSM 880 Contents ZEISS CHAPTER 4 ANNEX CONTENTS Page Software and Hardware Options ................. 2 Software ..........................2 Hardware ........................... 4 Courses on "How to Operate the System in an Optimized Way"...... 5 Beam Path Configuration Guide for Fluorescence Microscopy ......5 Optical Elements in the Configuration Control Window ............
Page 604: Software And Hardware Options
CHAPTER 4 - ANNEX ZEISS Software and Hardware Options LSM 880 Software and Hardware Options This section describes optional software and hardware configurations. Depending on your configuration, the content of dialogue and function may differ. Software The following software packages for ZEN 2 are available: −...
Page 605 CHAPTER 4 - ANNEX LSM 880 Software and Hardware Options ZEISS If your configuration does not include the "ZEN Module Visual Basic Macro, VBA", the following functions are not available: − New, Save and Save as buttons in the Macro Control window −...
Page 606: Hardware
CHAPTER 4 - ANNEX ZEISS Software and Hardware Options LSM 880 Hardware Depending on whether the following hardware components are available or not, the content of the screens may differ: − Piezo objective focusing device / Z-Piezo stage − X-Y scanning stage (different types available) −...
Page 607: Courses On "How To Operate The System In An Optimized Way
ZEISS Courses on "How to Operate the System in an Optimized Way" ZEISS is offering training courses on how to operate the system in an optimized way. − Courses are held in our application center in Jena, Germany. − Courses are held in English or German language, respectively.
Page 608: Setup Of Tracks Using Single Detectors (Channel Mode)
CHAPTER 4 - ANNEX ZEISS Beam path Configuration Guide for … LSM 880 Setup of Tracks Using Single Detectors (Channel Mode) a) One track only – simultaneous acquisition of channels • Switch on the suitable lasers for excitation of the dyes in the specimen in the Laser tool.
Page 609 CHAPTER 4 - ANNEX LSM 880 Beam path Configuration Guide for … ZEISS • Add a new track by clicking the button next to Tracks in the Imaging Setup tool. • Create a second track for AF 568. • By selecting the dyes from the pull-down menu, you can see the emission spectra to set the matching detection bands.
Page 610: Beam Path Configuration For Multi Photon Excitation
CHAPTER 4 - ANNEX ZEISS Beam path Configuration Guide for … LSM 880 Line and Frame Mode of Multitracking • Settings can be used for Line or Frame wise multi-tracking acquisition. • In Line mode the lines are scanned in turns for all tracks with the corresponding laser lines turned on exclusively.
Page 611: Recommendations For Excitation Laser Lines And Emission Filters Of Dyes
CHAPTER 4 - ANNEX LSM 880 Recommendations for Excitation Laser Lines and Emission Filters of Dyes ZEISS Recommendations for Excitation Laser Lines and Emission Filters of Dyes Laser line/MBS Emission bands DAPI > 385/420, max. at 461 EBFP > 385/420, max. at 447 Hoechst >...
Page 612: How To Access Data Acquired With Airyscan Using Other Software
The raw pixel data is contained in the CZI file in dimension H. If you can read CZI files you can directly access the Airyscan raw data channels. They have 32 entries in the H dimension. The documentation of the CZI file format can be requested via Zeiss web page: http://www.zeiss.com/microscopy/en_de/products/microscope-software/zen-2012/czi.html Additional Metadata is available in the "IAimImageTrackParameters"...
Page 613: Piezo Focusing Device
CHAPTER 4 - ANNEX LSM 880 Piezo Focusing Device ZEISS Piezo Focusing Device A Z-Piezo allows Z-stacks to be acquired considerably quicker compared to the focus drive of the microscope stand. The speed advantage depends on the total travel range in Z and the speed of the acquisition of the individual image of a Z-stack.
Page 614 CHAPTER 4 - ANNEX ZEISS Piezo Focusing Device LSM 880 Objectives: − M27 Installation: • Screw in your microscope objective into Piezo Objective Focusing Device (see Fig. 3/1). • Screw the thread-ring into your microscope (see Fig. 3/2). • Easy clamp the Piezo Objective Focusing Device on the thread-ring (see Fig.
Page 615: Specification Of Trigger-Interface
CHAPTER 4 - ANNEX LSM 880 Specification of Trigger-Interface ZEISS Specification of Trigger-Interface The LSM provide two ways to control external devices or be controlled via external devices. Synchronization signals: This interface is provided directly by the electronics of the system and cannot be influenced via the user inter face.
Page 616 CHAPTER 4 - ANNEX ZEISS Specification of Trigger-Interface LSM 880 Connectors: Pixelclock = Coax: Triax Lemo S. A. Serie 00, EPL.00.650NLN Signal name Description Signal PCLK_Out PixelClock Output Signal shield Digital ground Outer shield Shield Chassis ground The pixel clock is exactly symmetrical to the pixel. For example if the pixel rate is 10 kHz the high-level and the low level both have a time of 50 microseconds.
Page 617 CHAPTER 4 - ANNEX LSM 880 Specification of Trigger-Interface ZEISS 15-point high density D-Type plug: Plug "B" Signal name Description Hardware output only SyncOUT4 ** (stack) Synchronous output Sync. signal: Stack out SyncOUT5 out Grabbing Synchronous output Sync. signal: Grabbing...
Page 618 CHAPTER 4 - ANNEX ZEISS Specification of Trigger-Interface LSM 880 Type/Voltage Range: TTL signal level 3.3 V, CMOS low power consumption − 5.0 V tolerant input/output for interfacing with 5 V logic − Load: Output: < 50 mA (internal serial 68 ohm resistor) Input: 4.7 kOhm input impedance (internal 4.7 kOhm pullup to 3.3 V)
Page 619 CHAPTER 4 - ANNEX LSM 880 Specification of Trigger-Interface ZEISS User interface box for synchronization and trigger signals (optional) The interface box provides connections to the trigger IN and OUT functions accessible in the software user interface, the synchronization signals and the pixel clock. External devices can be connected to the LSM via standard BNC cables that are plugged into the user interface box.
Page 620: Level-Adjustable Sample Holder With Insert For Piezo Stage (Elyra)
CHAPTER 4 - ANNEX ZEISS Level-adjustable Sample Holder with Insert for Piezo Stage (ELYRA) LSM 880 Level-adjustable Sample Holder with Insert for Piezo Stage (ELYRA) Caution! The adjustment screws and other part of adjustable sample holders may collide with parts of the microscope, e.g. the condenser unit. When moving a motorized stage, avoid any collisions.
Page 621: Stage Leveling Using X-Cite In Reflection
CHAPTER 4 - ANNEX LSM 880 Level-adjustable Sample Holder with Insert for Piezo Stage (ELYRA) ZEISS Press the lower pointing arrow of the X input box of the Stage tool to return to the initial position. If the image is in focus (Fig. 6/F) X leveling is complete; otherwise repeat steps 3a) - e) iteratively.
Page 622 ZEISS Level-adjustable Sample Holder with Insert for Piezo Stage (ELYRA) LSM 880 Draw fiber plug of X-Cite completely out from the X-Cite casing. Start acquisition. The image will be dark since fiber was pulled out. Put fiber back and slowly move in until you will see a coarsely adjusted image to have sufficiently reduced light levels you can work with (Fig.
Page 623: Level-Adjustable Sample Holder For Lsm
CHAPTER 4 - ANNEX LSM 880 Level-adjustable Sample Holder for LSM ZEISS Level-adjustable Sample Holder for LSM Caution! Adjustable stage insert screws may intersect with parts of the microscope, e.g. the condenser unit. When moving a motorized stage, avoid any collisions. Do not install inserts while the stage is moving.
Page 624: Stage Leveling Using Lsm In Reflection Mode And Z-Piezo
ZEISS Level-adjustable Sample Holder for LSM LSM 880 3. Start a reflection scan at zoom 1.0x and focus on the border between coverslip and sample, which can be identified by the brightest reflection. A high pixel resolution is not important (use e.g. the 256x256 image format).
Page 625 CHAPTER 4 - ANNEX LSM 880 Level-adjustable Sample Holder for LSM ZEISS Start a reflection bidi line bidi z scan at zoom 1.0x and focus on the border between coverslip and sample, which can be identified by the brightest reflection line. A high pixel resolution is not important (use e.g.
Page 626: Liquid Cooling System Of The Lsm
Peltier cooling of the detectors (GaAsP PMT detectors as well one of the side multi-alkali PMTs). The liquid used for the device can be ordered via ZEISS service. The system is equipped with an optic and acoustic warning if it does not function as needed. The...
Page 627 CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 Contents ZEISS CHAPTER 5 MULTIPHOTON LASER SCANNING MICROSCOPY WITH LSM FAMILY SYSTEMS CONTENTS Page NLO (Non Linear Optics) Laser for LSM ............... 2 Laser Control tool ....................... 3 Imaging Setup and Channels tool ..................4 Motorized Periscope ......................
Page 628: Nlo (Non Linear Optics) Laser For Lsm
CHAPTER 5 - MULTIPHOTON LASER SCANNING … ZEISS NLO (Non Linear Optics) Laser for LSM LSM 880 NLO (Non Linear Optics) Laser for LSM The LSM scanning module is operated with a Titanium-Sapphire-Laser and optionally in combination with an optical parametric oscillator (OPO) to perform Multiphoton Microscopy. Depending on the type of laser the following operation procedures apply: •...
Page 629: Laser Control Tool
CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 NLO (Non Linear Optics) Laser for LSM ZEISS Laser Control tool The Laser Control tool shows the NLO laser as an additional laser of the system. Operation of the Chameleon (Coherent), OPO and Mai Tai (Spectra Physics) laser •...
Page 630: Imaging Setup And Channels Tool
CHAPTER 5 - MULTIPHOTON LASER SCANNING … ZEISS NLO (Non Linear Optics) Laser for LSM LSM 880 Imaging Setup and Channels tool • Setting the wavelength of the laser/OPO • The wavelength of the laser and OPO can be set in the Imaging Setup tool in the pop up window of the laser icon or in the Channels tool.
Page 631: Motorized Periscope
CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 NLO (Non Linear Optics) Laser for LSM ZEISS Motorized Periscope • Systems with OPO or two lasers coupled into one scan head are equipped with a motorized periscope. The interface for controlling the motorized mirrors within the periscope is in the Maintain tab/System Options tool.
Page 632: Instructions For Beam Adjustment Using The Motorized Periscope
CHAPTER 5 - MULTIPHOTON LASER SCANNING … ZEISS NLO (Non Linear Optics) Laser for LSM LSM 880 1.3.1 Instructions for beam adjustment using the motorized periscope Your system is equipped with a periscope that provides motorised mirrors for the guidance of two beams of light of either two NLO lasers or one NLO laser and one OPO.
Page 633 CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 NLO (Non Linear Optics) Laser for LSM ZEISS Motorized Periscope Panel This panel provides the interface to control the motorized mirrors in the periscope Beam 1 and Beam 2 represent the two input beams according to the following constellation:...
Page 634 CHAPTER 5 - MULTIPHOTON LASER SCANNING … ZEISS NLO (Non Linear Optics) Laser for LSM LSM 880 The following procedures describe how to adjust positioning and illumination for the acquired images using the ZEN software. 1.3.1.1 Correction of the illumination profile The illumination profile indicates whether there is an angular offset of the beam when scanning the sample.
Page 635 CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 NLO (Non Linear Optics) Laser for LSM ZEISS Horizontal shift of the image generated with Beam1 (NLO laser) to the right (To shift the image horizontally to the left, just use the reverse direction buttons): 1.
Page 636 CHAPTER 5 - MULTIPHOTON LASER SCANNING … ZEISS NLO (Non Linear Optics) Laser for LSM LSM 880 Vertical shift of the image generated with Beam1 (NLO laser) downwards (To shift the image upwards, just use the reverse direction buttons): 1. Click the right "fine movement" button of Beam1/Output/Axis1 repeatedly until an image shift and illumination change is visible (image gets darker).
Page 637: Pinhole And Collimator Settings
CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 NLO (Non Linear Optics) Laser for LSM ZEISS Pinhole and Collimator Settings • The pinhole can be fully opened for maximum detection efficiency due to the intrinsic optical sectioning capabilities of the NLO effect (see Channels tool).
Page 638: Non Descanned Detection (Ndd)
LSM 880 Non Descanned Detection (NDD) The use of Non-Descanned Detectors with the LSM 880 NLO is only useful in combination with the NLO laser. The Non-Descanned Detection modules can be used on the reflected or transmitted-light beam path (transmission NDDs not available for Axio Imager) or simultaneously on both beam paths.
Page 639: Mounting And Dismounting Of Ndd Modules From The Ndd Port
CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 Non Descanned Detection (NDD) ZEISS Mounting and Dismounting of NDD modules from the NDD port To take off a NDD module from its port or from the NDD module before loosen the screws (Fig. 6/1 und 2) on the port connection.
Page 640 The NDD detectors need to be connected to the systems in the following way: LSM 880 NLO LVDS A (LSM 880 detection) LVDS B T-PMT (via LSM 880 scanner) LVDS C Reflected light NDDs (followed by NDD module channel 5, followed by GaAsP NDD 2 channels) LVDS D BiG.2 on DCout port followed by Transmitted light NDDs (followed by NDD...
Page 641 CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 Non Descanned Detection (NDD) ZEISS In addition the power connections have to be made in the following way to the plugs on the back of the electronics rack: Fig. 9 Power connections of NDD detectors and the scan modules on the electronics rack of the LSM NLO.
Page 642: Filter For Non Descanned Detectors (Ndds)
(maximum of two channels is possible). All dichroic filters offered by ZEISS for this modules, are long pass dichroic filters. Therefore the blue part of the spectrum is detected with the first NDD whereas the red part is guided further on to the following NDDs (Fig. 11).
Page 643 CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 Non Descanned Detection (NDD) ZEISS Fig. 11 Example for a filter equipment of a four channel NDD set up on Axio Observer starting with an NDD module type C or D For LSM NLO systems on Axio Imager (NDDs are only available for reflection) and Axio Examiner a total of 5 detectors can be mounted onto the ports starting with a two channel module of type A or B.
Page 644 ZEISS Non Descanned Detection (NDD) LSM 880 For example, to add a fourth channel to the cascade shown in Fig. 13 with a filter combination to additionally detect an emission signal in the far red range the filters have to be rearrange the following way: Fig.
Page 645 CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 Non Descanned Detection (NDD) ZEISS For Axio Examiner and Axio Imager: BiG.2 with GaAsP at Reflected Light NDD Port BiG NDD as channel 1+2 NDD module type A, 2 channels for upright...
Page 646 CHAPTER 5 - MULTIPHOTON LASER SCANNING … ZEISS Non Descanned Detection (NDD) LSM 880 BiG NDD as channel 1+2 NDD module type C, 2 channels for inverted microscopes, BiG NDD module as channel 3+4 BiG.2 with GaAsP at Transmitted Light NDD Port External detection module BiG DC/transmitted light BiG NDD as channel 1+2;...
Page 647 CHAPTER 5 - MULTIPHOTON LASER SCANNING … LSM 880 Non Descanned Detection (NDD) ZEISS GaAsP NDD 2 channels for Axio Examiner This detector module is located in a special objective holder. It replaces a standard objective holder on the Axio Examiner. It holds one objective. Standard objectives need to be used together with the optical adapter provided.
Page 648: Changing The Filter Modules Big.2
CHAPTER 5 - MULTIPHOTON LASER SCANNING … ZEISS Non Descanned Detection (NDD) LSM 880 Changing the Filter Modules BiG.2 You can remove and replace filter modules by push and click. When opening the filter insert, the safety control will switch off the laser light.
Page 649 Carl Zeiss Confocal Laser Scanning Microscopy...
Page 651 Confocal Laser Scanning Microscopy Stefan Wilhelm Carl Zeiss Microscopy GmbH · Carl-Zeiss-Promenade 10 · 07745 Jena · Germany...
Page 652 Page Introduction PART 1 OpticalImageFormation Part 1 deals with the purely optical conditions in a confocal LSM and the influence of the pin - hole on image formation. From this, ideal values for resolution and optical slice thickness are derived. PointSpreadFunction ResolutionandConfocality Resolution...
Page 653 In recent years, the confocal Laser Scanning Microscope (LSM) has become widely established as a research instrument. The present brochure aims at giving a scientifically sound survey of the special nature of image formation in a confocal LSM.
Page 654 Introduction Imagegeneration Following a description of the fundamental differences between a conventional and a confocal microscope, this monograph will set out the special features of the confocal The complete generation of the two-dimensional object LSM and the capabilities resulting from them. information from the focal plane (object plane) of a confo- The conditions in fluorescence applications will be given cal LSM essentially comprises three process steps:...
Page 655 The descriptions below exclusively cover the point scanner principle as implemented, for example, in laser scanning microscopes from Carl Zeiss. Configurations in which several ob- Fig. 2 Beam path in a confocal LSM. A microscope objective lens is used to focus a laser beam onto the specimen, where it excites fluorescence, for ject points are irradiated simultaneously are not considered.
Page 656 Opticalslices Hence it follows that a confocal LSM can be used to advan- tage especially where thick fluorescent specimens (such as With a confocal LSM and it's variable pinhole it is therefore biological cells in tissue) have to be examined. The possibil- possible to exclusively image a thin optical slice out of a ity of optical sectioning eliminates the drawbacks attached thick specimen (typically, up to 100 µm), a method known...
Page 657 dimension Timeseries In addition to the possibility to observe a single plane (or A further field of importance is the investigation of living slice) of a thick specimen in good contrast, optical section- specimens that show dynamic changes even in the range of ing allows a great number of slices to be cut and recorded at microseconds.
Page 658 Point Spread Function In order to understand the optical performance charac- teristics of a confocal LSM in detail, it is necessary to have a closer look at the fundamental optical phenomena re- sulting from the geometry of the confocal beam path. As mentioned before, what is most essential about a confocal LSM is that both illumination and observation (detection) are limited to a point.
Page 659 OpticalImageFormation PART 1 The total PSF (PSF ) of a confocal microscope behind the is also influenced by all these factors and, addition- pinhole is composed of the PSFs of the illuminating beam ally, by the pinhole size. For reasons of beam path efficiency path (PSF ;...
Page 660 Resolution and Confocality Wherever quantitative data on the resolving power and The smaller the pinhole diameter, the more PSF depth discrimination of a confocal LSM are specified, it is proaches the order of magnitude of PSF . In the limit case necessary to distinguish clearly whether the objects they (PH <...
Page 661 OpticalImageFormation PART 1 Resolution Resolution, in case of large pinhole diameters (PH >1AU), is meant to express the separate visibility, both laterally and axially, of points during the scanning process. Imagine an object consisting of individual points: all points spaced seite 7 closer than the extension of PSF are blurred (spread), i.e.
Page 662 Let the statements made on the PSF so far be further illus- Optical axis trated by the figure on the left. It shows a section through the resulting diffraction pattern surrounding the focus on the illumination side (PSF The lines include areas of equal brightness (isophote pre- sentation).
Page 663 seite 7 OpticalImageFormation PART 1 (x,y,z) = PSF (x,y,z) (x,y,z) seite 9 0.88 . FWHM Geometricopticalconfocality Likewise, in the case of geometric-optical confocality, there ill,axial (n- n is a linear relationship between depth discrimination and Optical slice thickness (depth discrimination) and stray light pinhole diameter.
Page 664 seite 11 0.88 . seite 11 FWHM ill,axial (n- n 0.88 . seite 9 2 . n . PH 0.88 . FWHM 2 . n . PH det,axial FWHM n- n det,axial n- n 0.88 . seite 12 FWHM 1.77 . n . ill,axial (n- n (x,y,z) =...
Page 665 OpticalImageFormation PART 1 From equations (7) and (7a) it is evident that depth resolu- From the applications point of view, the case of strictly tion varies linearly with the refractive index n of the immer- wave-optical confocality (PH=0) is irrelevant (see also Part 2). sion liquid and with the square of the inverse value of the By merely changing the factors in equations (7) and (8) it numerical aperture of the objective lens {NA = n ·...
Page 666 Optical slice (NA = 1.3; n = 1.52; λ  = 496 nm) Fig. 11 a) Variation of pinhole diameter Pinhole diameter [AU] 1000 Depth resolution b) Variation of (PH = 0; n = 1.52; numerical aperture λ  = 496 nm) Numerical aperture Depth resolution c) Variation of...
Page 667 OpticalImageFormation PART 1 Overview seite 15 0.88 . 0.64 . 2 . n . PH (n- n n- n Conventional microscopy Confocal microscopy 1 AU < PH <∞ Confocal microscopy PH < 0.25 AU not definable 1. Optical slice thickness1) 1.
Page 668 Sampling and Digitization After the optical phenomena have been discussed in Part 1, Part 2 takes a closer look at how the digitizing process and system-inherent sources of noise limit the performance of the system . As stated in Part 1, a confocal LSM scans the specimen surface point by point.
Page 669 (e.g. fluorescence), this is a decisive advantage over point sampling with regard to the signal-to-noise ratio (SNR). Therefore, Carl Zeiss confocal LSM systems operate in the integration mode. The absolute integration time can be modified by varying the scanning speed, which also means Fig.
Page 670 Nyquisttheorem If the number of sampling points per feature size is smaller than that given by the Nyquist theorem (undersampling), It is known from Part 1 that the information content of the part of the information will be lost. This is evident in signal is limited by the resolving power of the microscope Figure 14c especially by the unresolved fine features.
Page 671 (see the example below). length of the scanned field by a factor of 2) 3.92 system constant In this way, the user of a ZEISS confocal LSM can control the Z ≥ Magnification objective magnification...
Page 672 Noise The main types of noise important in a confocal LSM system With a pinhole diameter <1AU, resolution improves (better are detector noise (dark noise, secondary emission noise), point separation thanks to a deeper dip), which is penalized laser noise, and shot noise of the light (see Details “Sources by a drastic loss in energy.
Page 673 SignalProcessing PART 2 Resolutionandshotnoise– Figure 17 (page 24) shows the dependence of the resolu- tion probability on signal level and pinhole diameter by the resolutionprobability example of a two-point object and for different numbers of If the number of photons detected (N) is below 1000, fluo- photoelectrons per point object.
Page 674 A resolution probability of 90% is considered ne cessary for The pinhole diameter selected in practice will therefore al- resolving the two point images. Accordingly, the two-point ways be a trade-off between two quality parameters: noise object defined above can only be resolved if each point (SNR as a function of the intensity of the detected light) produces at least about 25 photoelectrons.
Page 675 SignalProcessing PART 2 PossibilitiestoimproveSNR pixel remains constant. Photon statistics are improved by the addition of photons from several scanning runs (SNR Pinhole diameters providing a resolution probability below =  n·N  ; N = constant, n = number of scans averaged). By Variation of pixel time 90% may still yield useful images if one uses a longer pixel comparison, a longer pixel time directly improves the pho-...
Page 676 The pictures on the left demonstrate the influence of pixel time and averaging on SNR; object details can be made out much better if the pixel time increases or averaging is employed. Another sizeable factor influencing the SNR of an image is the efficiency of the detection beam path.
Page 677 Glossary Aperture angle of a microscope objective Airy unit (diameter of Airy disc) dpix Pixel size in the object plane FWHM Full width at half maximum of an intensity distribution (e.g. optical slice) Refractive index of an immersion liquid Numerical aperture of a microscope objective Pinhole;...
Page 679 Details PART 3 To give some further insight into Laser Scanning Microscopy, the following pages treat several aspects of particular importance for practical work with a Laser Scanning Microscope. Pupil Illumination Optical Coordinates Fluorescence Sources of Noise...
Page 680 Airy function (also known tion of the illuminating beam cross-section at the pupil as Airy disk) [In Carl Zeiss microscope objectives, the pupil plane causes a certain energy loss and thus a decrease in diameter is implemented by a physical aperture close to the efficiency.
Page 681 Details PART 3 Optical Coordinates In order to enable a representation of lateral and axial quan- Analogously, a sensible way of normalization in the axial tities independent of the objective lens used, let us intro- direction is in terms of multiples of the wave-optical depth duce optical coordinates oriented to microscopic imaging.
Page 682 Fluorescence Fluorescence is one of the most important contrasting In principle, the number of photons emitted increases with methods in biological confocal microscopy. the intensity of excitation. However, the limiting parameter Cellular structures can be specifically labeled with dyes is the maximum emission rate of the fluorochrome mole- (fluorescent dyes = fluorochromes or fluorophores) in vari- cule, i.e.
Page 683 Details PART 3 What has been said so far is valid only as long as the mol- 1.5 . 10 ecule is not affected by photobleaching. In an oxygen-rich environment, fluorescein bleaches with a quantum efficien- 1.29 . 10 cy of about 2.7·10 .
Page 684 seite II 1.22 seite II 1.22 1AU = 1AU = 1.22 seite II 1AU = 1RU = 1RU = Sources of Noise 1RU = Sources of noise effective in the LSM exist everywhere in the Darknoise signal chain – from the laser unit right up to A/D conver- Dark noise is due to the generation of thermal dark elec- sion.
Page 685 Literature 1.Beyer,H., 11.Pawley,J., Handbuch der Mikroskopie, 2nd Edition, Handbook of Biological Confocal Microscopy, VEB Verlag Technik Berlin, (1985) Springer, 3rd Edition (2006) 2.Born&Wolf, 12.Stelzer,E.H.K., Priniples of Optics, The intermediate optical system of laser scanning 6th edition 1988, Pergamon Press confocal microscopes; Handbook of Biological Confocal Microscopy, pp.
Page 686 We gratefully acknowledge the assistance of Bernhard Gröbler, Hartmut Heinz and all other staff members who contributed to this brochure. Carl Zeiss Microscopy GmbH 07740 Jena, Germany BioSciences | Jena Location Phone: +49 3641 64 3400 Telefax: +49 3641 64 3144 E-Mail: micro@zeiss.de...
Page 689 Highlights of Laser Scanning Microscopy 1982 The first Laser Scanning Microscope from Carl Zeiss. The prototype of the LSM 44 series is now on display in the Deutsches Museum in Munich. 1988 The LSM 10 – a confocal system with two fluorescence channels.
Page 690 This monograph comprehensively deals with the quality parameters of resolution, depth discrimination, noise and digitization, as well as their mutual interaction. The set of equations presented allows in-depth theoretical investigations into the feasibility of carrying out intended experiments with a confocal LSM.

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