Page 1 LSM 510 Laser Scanning Microscope Operating Manual...
Page 2 INTRODUCTION LSM 510 Knowledge of this manual is required for the operation of the instrument. Would you therefore please make yourself familiar with the contents of this manual and pay special attention to hints concerning the safe operation of the instrument.
Page 3 INTRODUCTION LSM 510 Developed in Collaboration with the European Molecular Biology Laboratory (EMBL) PF 102209 Meyerhofstr. 1 D-69012 Heidelberg Phon: ++49-(0)-62221-387-0 Fax: ++49-(0)-62221-387-306 B 40-051 e 07/98...
Page 4 INTRODUCTION LSM 510 B 40-051 e 07/98...
Page 5 INTRODUCTION LSM 510 How to make best use of the LSM 510 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 6: Table Of Contents
..........Axioplan 2 MOT 000000-1028-778 Axiovert 100 M SP 000000-1028-779 Axiovert 100 M BP 000000-1028-780 Objectives: ............................Confocal Laser Scanning Module LSM 510 Configuration 1 000000-1027-076 Configuration 2 000000-1027-077 Configuration 3 000000-1027-078 Configuration 4 000000-1027-079 Configuration 5 000000-1027-080...
Page 7 000000-1027-823 Filter changer 18 mm 453070-0000-000 Set of INDO filters 447960-0000-000 Set of SNARF filters 447961-0000-000 The LSM 510 in the configuration as checked above was installed and handed to the customer in functional condition ....................Phone: ..........Fax: ..........
Page 8 The Setup Requirements section outlines the installation and supply requirements of the LSM 510 Microscope System, together with the relevant specifications. Here you will find an introduction to Laser Scanning Microscopy, with an explanation of the principles of confocal imaging.
Page 9 INTRODUCTION LSM 510 Notes on Device Safety LSM 510 Setup Requirements Introduction to Laser Scanning Microscopy Operation Software 3D Description B 40-051 e 07/98...
Page 10 INTRODUCTION LSM 510 B 40-051 e 07/98...
Page 11 NOTES ON DEVICE SAFETY LSM 510 Contents CHAPTER 1 NOTES ON DEVICE SAFETY CONTENTS NOTES ON DEVICE SAFETY ...................1-3 General ...........................1-3 Regulations ........................1-3 Notes on setting up the microscope system..............1-4 Notes on handling the computer and data media .............1-5 Notes on care, maintenance and service................1-6 Notes on handling the laser components................1-7...
Page 12 NOTES ON DEVICE SAFETY Contents LSM 510 B 40-051 e 07/98...
Page 13: Notes On Device Safety
The manufacturer will not assume liability for any malfunction or damage caused by any thing other than the intended use of the LSM 510 or individual modules or parts of it, nor by any repair or other service operation performed or attempted by persons other than duly authorized service staff.
Page 14: Notes On Setting Up The Microscope System
Crate 6: small system table The LSM 510 must be set up so as to ensure that the minimum clearance between the wall and the rear of the system is no less than 0.5 m. This clearance is needed for adjustment and maintenance operations.
Page 15: Notes On Handling The Computer And Data Media
NOTES ON DEVICE SAFETY LSM 510 Notes on handling the computer and data media Notes on handling the computer and data media The computer used as standard in your LSM system is an IBM-compatible high-end pentium computer with WINDOWS NT (Version 4.0) operating system.
Page 16: Notes On Care, Maintenance And Service
To complete cleaning, lightly breathe on the glass surface and rub it dry with a clean cloth. Lint or dust is best removed with a clean hairbrush. The air filter mat at the bottom of the LSM 510 Control Unit must be cleaned every six months. Filter mats can be ordered from our Service Department.
Page 17: Notes On Handling The Laser Components
If used properly, the LSM 510 will not pose any laser radiation risks for operating staff. The dangerous laser radiation area is limited to the beam path and to a distance of up to around 10 cm from the specimen.
Page 18: Warning And Information Labels
The warning and information labels attached on the LSM 510 must be observed. Check whether all of the labels shown below are provided on your instrument, and contact Carl Zeiss Germany or one of the service agencies if you should discover that any of the labels should be missing.
Page 19 NOTES ON DEVICE SAFETY LSM 510 Warning and information labels Fig. 1-2 Warning and information labels on the Axioplan 2 microscope with LSM 510 scanning module B 40-051 e 07/98...
Page 20 NOTES ON DEVICE SAFETY Warning and information labels LSM 510 WARNING: LASER RADIATION Avoid exposure to beam when cover is removed Carl Zeiss LASERMODUL POWER REMOTE CONTROL AOTF 230V/ POWER 2 REMOTE SAFETY 2x400VA LASER F3/T3,15A-H POWER 3 POWER 1 230V/1300VA...
Page 21 NOTES ON DEVICE SAFETY LSM 510 Warning and information labels BEAM AVOID EXPOSURE OPEN CLOSED BEAM LASER RADIATION IS EMITTED OPEN CLOSED FROM THIS APERTURE Laser - Fertigung GmbH TATZENPROMENADE 1a D-07745 JENA LGK 7774 Q4001-K7784 S.NO.000 MADE IN GERMANY...
Page 22 NOTES ON DEVICE SAFETY Warning and information labels LSM 510 1-12 B 40-051 e 07/98...
Page 23 LSM 510 - SETUP REQUIREMENTS LSM 510 Contents CHAPTER 2 LSM 510 - SETUP REQUIREMENTS CONTENTS LSM 510 - SETUP REQUIREMENTS................2-3 Space Requirements....................2-3 2.1.1 LSM (one microscope, large system table): 320 cm x 220 cm ........2-3 2.1.2 LSM with Ar laser (UV): 340 x 260 cm .................2-3 2.1.3...
Page 24 LSM 510 - SETUP REQUIREMENTS Contents LSM 510 B 40-051 e 07/98...
Page 25: Lsm 510 - Setup Requirements
LSM 510 - SETUP REQUIREMENTS LSM 510 Space Requirements LSM 510 - SETUP REQUIREMENTS Space Requirements 2.1.1 LSM (one microscope, large system table): 320 cm x 220 cm Fig. 2-1 2.1.2 LSM with Ar laser (UV): 340 x 260 cm We recommend placing the cooling unit of the Ar laser (UV) in a separate room to prevent heat accumulation and vibration.
Page 26: Lsm With Ar Laser (Uv) And Two Microscopes: 450 X 220 Cm
LSM 510 - SETUP REQUIREMENTS Space Requirements LSM 510 2.1.3 LSM with Ar laser (UV) and two microscopes: 450 x 220 cm We recommend placing the cooling unit of the Ar laser (UV) in a separate room to prevent heat accumulation and vibration. Length of the water hose: 400 cm.
Page 27: Power Requirements
Power Requirements Power Requirements The LSM 510 comes with a mains power supply cord and plug, either CEE red (230 V, 16 A, 3 phases), or CEE yellow (115 V, 32 A, 3 phases), and with the matching mains socket outlet.
Page 28: Phase 1 (Lsm)
LSM 510 - SETUP REQUIREMENTS Power Requirements / Physical Dimensions LSM 510 2.2.1 Phase 1 (LSM) feeds the following units: Laser Module HeNe 2x via Power 1 (5-socket adapter) Computer + monitor Microscope MCU 28 Scanning Module via Power 2:...
Page 29: Dimension Of Shipment Crates
LSM 510 - SETUP REQUIREMENTS LSM 510 Dimensions ... / Envirenment Requirements / Vibrations Dimension of shipment crates Crate containing Length (cm) Width (cm) Height (cm) Weight (kg) Large system table Small system table Monitor, computer UV laser unit UV cooling unit...
Page 30: Laser Specifications
LSM 510 - SETUP REQUIREMENTS Laser Specifications LSM 510 Laser Specifications 2.7.1 Coherent Enterprise 653 II: 352, 364 nm, 80 mW, laser power class 3 B Line voltage 100...240 V Line frequency 50...60 Hz Max. current 1 phase at 32...63 A...
Page 31: Lasos Lgk 7812 Ml-1/Lgn 7812: 458, 488, 514 Nm, 25 Mw, Laser Power Class 3 B
LSM 510 - SETUP REQUIREMENTS LSM 510 Dimensions ... / Envirenment Requirements / Vibrations 2.7.4 LASOS LGK 7812 ML-1/LGN 7812: 458, 488, 514 nm, 25 mW, laser power class 3 B Line voltage 100...240 V Line frequency 50...60 Hz Max. current...
Page 32: Microscopes
LSM 510 - SETUP REQUIREMENTS Laser Specifications LSM 510 Microscopes Inverted Axiovert 100 M BP or SP Upright Axioplan 2 MOT All Zeiss ICS objectives and accessories can be accommodated. Z motor DC servomotor, opto-electronically coded Least Z interval: 100 nm HRZ-200 Galvanometer-driven precision focusing stage Max.
Page 33: Laser Module Vis
LSM 510 - SETUP REQUIREMENTS LSM 510 Microscopes / Scanning Module 2.10 Laser Module VIS Single-mode polarization preserving fiber Laser beam attenuation for all lasers by VIS-AOTF HeNe laser (543 nm, 1 mW) HeNe laser (633 nm, 5 mW) Ar laser (458, 488, 514 nm, 25 mW)
Page 34 LSM 510 - SETUP REQUIREMENTS Laser Module VIS / Laser Module UV LSM 510 2-12 B 40-051 e 07/98...
Page 35 CONTENTS INTRODUCTION TO LASER SCANNING MICROSCOPY ..........3-3 Principle of Laser Scanning Microscopy ................3-3 Three-Dimensional Presentations of LSM Image Stacks..........3-4 Optical Diagram of the LSM 510 (Schematic) ...............3-6 Performance Features of the LSM 510 .................3-7 3.4.1 Optical and mechanical aspects ...................3-7 3.4.2...
Page 36 INTRODUCTION TO LASER SCANNING MICROSCOPY Contents LSM 510 B 40-051 e 07/98...
Page 37: Introduction To Laser Scanning Microscopy
INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Principle of Laser Scanning Microscopy INTRODUCTION TO LASER SCANNING MICROSCOPY 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 38: Three-Dimensional Presentations Of Lsm Image Stacks
INTRODUCTION TO LASER SCANNING MICROSCOPY Three-Dimensional Presentation of LSM Image Stacks LSM 510 In order to obtain an image of the selected object plane as a whole, it is necessary to scan the object plane in a point-by-point, line-by-line raster by means of an XY light deflection system. The detectors - as a rule, photomultipliers - convert the optical information into electric signals.
Page 39 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Three-Dimensional Presentation of LSM Image Stacks Orthogonal sections This computation produces a triplet of mutually perpendicular sectional images. Oblique sections A section through the stack is made along an oblique plane defined by the selection of five coordinates, i.e.
Page 40: Optical Diagram Of The Lsm 510 (Schematic)
INTRODUCTION TO LASER SCANNING MICROSCOPY Optical Diagram of the LSM 510 LSM 510 Optical Diagram of the LSM 510 (Schematic) Mirror VIS Fiber Collimator UV Fiber LSF NDF Monitor Eyepiece Plate Diode Scan Pinhole Fiber Coupler Tube Lens Lens Optics...
Page 41: Performance Features Of The Lsm 510
INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Performance Features of the LSM 510 Performance Features of the LSM 510 3.4.1 Optical and mechanical aspects The highly integrated system design makes for the shortest possible optical paths, top-grade optical precision and high stability. The compact scanning module can be fitted to an inverted (Axiovert 100 M BP or SP) or upright (Axioplan 2 ) microscope in less than three minutes.
Page 42: Microscope Equipment Of The Lsm 510 System
This will absolutely prevent "bleeding". 3.4.2 Microscope equipment of the LSM 510 system The LSM 510 system is equipped either with the Axiovert 100 M BP or SP microscope which is founded on the Axiovert 100/135 microscope serie, or with the Axioplan 2 microscope.
Page 43 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Performance Features of the LSM 510 e) The analyzer slider for conventional DIC methods will be operated from the right side and is located just below the nosepiece. When the rod is pushed in, the analyzer is located in the beam path. In LSM-mode the analyzer must not be located in the beam path, analyzer rod must be pulled out.
Page 44 INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 510 LSM 510 (3) Transmitted-light illumination a) The illuminator support contains a security circuit, which activates a shutter preventing laser light from reaching the stand when the support is moved to back. A complementary shutter built-in the stand prevents laser light from reaching the eye pieces during scanning mode.
Page 45: Computer Hardware And Software
The software of the LSM 510 has two levels. On the simple operator interface level, a result will be achieved after a few prompts; graphical prompting of the user in conjunction with automatic setting of many parameters is an ideal tool for daily routine jobs.
Page 46 INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 510 LSM 510 3-12 B 40-051 e 07/98...
Page 47 CONTENTS INTRODUCTION TO LASER SCANNING MICROSCOPY ..........3-3 Principle of Laser Scanning Microscopy ................3-3 Three-Dimensional Presentations of LSM Image Stacks..........3-4 Optical Diagram of the LSM 510 (Schematic) ...............3-6 Performance Features of the LSM 510 .................3-7 3.4.1 Optical and mechanical aspects ...................3-7 3.4.2...
Page 48 INTRODUCTION TO LASER SCANNING MICROSCOPY Contents LSM 510 B 40-051 e 07/98...
Page 49: Introduction To Laser Scanning Microscopy
INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Principle of Laser Scanning Microscopy INTRODUCTION TO LASER SCANNING MICROSCOPY 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 50: Three-Dimensional Presentations Of Lsm Image Stacks
INTRODUCTION TO LASER SCANNING MICROSCOPY Three-Dimensional Presentation of LSM Image Stacks LSM 510 In order to obtain an image of the selected object plane as a whole, it is necessary to scan the object plane in a point-by-point, line-by-line raster by means of an XY light deflection system. The detectors - as a rule, photomultipliers - convert the optical information into electric signals.
Page 51 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Three-Dimensional Presentation of LSM Image Stacks Orthogonal sections This computation produces a triplet of mutually perpendicular sectional images. Oblique sections A section through the stack is made along an oblique plane defined by the selection of five coordinates, i.e.
Page 52: Optical Diagram Of The Lsm 510 (Schematic)
INTRODUCTION TO LASER SCANNING MICROSCOPY Optical Diagram of the LSM 510 LSM 510 Optical Diagram of the LSM 510 (Schematic) Mirror VIS Fiber Collimator UV Fiber LSF NDF Monitor Eyepiece Plate Diode Scan Pinhole Fiber Coupler Tube Lens Lens Optics...
Page 53: Performance Features Of The Lsm 510
INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Performance Features of the LSM 510 Performance Features of the LSM 510 3.4.1 Optical and mechanical aspects The highly integrated system design makes for the shortest possible optical paths, top-grade optical precision and high stability. The compact scanning module can be fitted to an inverted (Axiovert 100 M BP or SP) or upright (Axioplan 2 ) microscope in less than three minutes.
Page 54: Microscope Equipment Of The Lsm 510 System
This will absolutely prevent "bleeding". 3.4.2 Microscope equipment of the LSM 510 system The LSM 510 system is equipped either with the Axiovert 100 M BP or SP microscope which is founded on the Axiovert 100/135 microscope serie, or with the Axioplan 2 microscope.
Page 55 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Performance Features of the LSM 510 e) The analyzer slider for conventional DIC methods will be operated from the right side and is located just below the nosepiece. When the rod is pushed in, the analyzer is located in the beam path. In LSM-mode the analyzer must not be located in the beam path, analyzer rod must be pulled out.
Page 56 INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 510 LSM 510 (3) Transmitted-light illumination a) The illuminator support contains a security circuit, which activates a shutter preventing laser light from reaching the stand when the support is moved to back. A complementary shutter built-in the stand prevents laser light from reaching the eye pieces during scanning mode.
Page 57: Computer Hardware And Software
The software of the LSM 510 has two levels. On the simple operator interface level, a result will be achieved after a few prompts; graphical prompting of the user in conjunction with automatic setting of many parameters is an ideal tool for daily routine jobs.
Page 58 INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 510 LSM 510 3-12 B 40-051 e 07/98...
Page 59 OPERATION LSM 510 Contents CHAPTER 4 OPERATION CONTENTS Page OPERATION ........................4-3 General ...........................4-3 Software .........................4-3 4.2.1 Boot WINDOWS NT ......................4-4 4.2.2 Log on to WINDOWS NT....................4-5 Quick start........................4-7 4.3.1 Starting the LSM Program ....................4-7 4.3.2 Creating a data base for image storage................4-8 4.3.3...
Page 60 OPERATION Contents LSM 510 Page Image optimization......................4-81 4.7.1 Detector Gain/Ampl. Offset/Ampl. Gain ................. 4-85 4.7.2 Pinhole adjustment......................4-89 4.7.3 Scan speed........................4-93 4.7.4 Channel Shift Function ....................4-98 Analysis of Images and Stacks..................4-101 Data base / Loading and Storing of images ..............4-138 4.9.1...
Page 61: Operation
It is based on the network-capable graphic 32-bit Microsoft ® WINDOWS NT 4.0 operating system. Portions ©Copyright 1996, Microsoft Corporation. All rights reserved. The installation of the software for the LSM 510 and the basic settings of the equipment components are exclusively carried out by Carl 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 62: Boot Windows Nt
OPERATION Software LSM 510 4.2.1 Boot WINDOWS NT Drive "A" must not contain a diskette. Normally, the LSM system is turned on with the REMOTE CONTROL switch. If this switch is not used, turn the system on with the "I" button on the laser module; in addition, the jumper plug supplied must be plugged at the POWER REMOTE CONTROL terminal.
Page 63: Log On To Windows Nt
OPERATION LSM 510 Software WINDOWS NT operating system is being loaded. The "Begin Logon" WINDOWS dialog box appears on the monitor. Fig. 4-2 4.2.2 Log on to WINDOWS NT Press the three keys <Ctrl>, <Alt> and <Del> at the same time.
Page 64 (Parfocal Settings) Fig. 4-4 Start LSM 510 dummy: A program that starts the LSM in only dummy mode. Useful in case of working with stored images with a separate workstation or when demonstrating the system without microscope, scan module, electronic box and laser module.
Page 65: Quick Start
From the LSM 510 Switchboard menu, click on the <Scan New Images> button and <Start Expert Mode> button. The LSM will go through a CP initialization and open a toolbar labeled LSM 510 expert mode. This tool bar LSM 510 - Expert Mode appears on the screen.
Page 66: Creating A Data Base For Image Storage
Creating a data base for image storage Fig. 4-6 Click on the <File> button from LSM 510 tool bar. Click on the <New> button. This will allow you to create a new data base to store your images, experimental setup and comments from your confocal session.
Page 67: Turning The Lasers On
Turning the Lasers On Fig. 4-8 Click on the <Acquire> button from the LSM 510 tool bar. Click on the <Laser> button. You will see a Laser-Control menu with a list of available lasers. Using the mouse, click on the laser(s) which has the appropriate wavelength to excite the dyes labeling your specimen.
Page 68: Look In The Microscope And Visually Set Up Your Specimen
4.3.4 Look in the Microscope and Visually Set Up Your Specimen Click on the <Micro> and <VIS> buttons from the LSM 510 tool bar. The Axioplan Control menu or the Axiovert Control menu appears on the screen. Dialog unit for Microscope Axioplan Fig.
Page 69 OPERATION LSM 510 Quick start Dialog unit for Microscope Axiovert Fig. 4-11 Move the silver slider on the side of the microscope to the appropriate position - the correct position will be shown in a message box on the monitor .
Page 70: Setting The Beam Path
After the specimen is focused and the area of interest is selected, close the menu and click on the <LSM> button from the LSM 510 tool bar. The software will direct you regarding the appropriate setting of the silver slider. If required, push silver slider to the correct position.
Page 71 OPERATION LSM 510 Quick start Click on the <Recording Configurations> button in the "Configuration Control" window. The "Recording Configurations" window appears on the screen. Fig. 4-13 A list of configurations will appear by clicking on the button. Choose a configuration from the list based on what you need to image from your specimen (e.g.
Page 72: Laser Scanning
LSM 510 4.3.6 Laser Scanning Click on the <Scan> button from the LSM 510 tool bar. The "Scan Control" window appears on the screen. Fig. 4-14 Click on the <Find> button on the right side of the Scan Control menu.
Page 73 OPERATION LSM 510 Quick start Click on the <Single> button on the right side of the Scan Control menu. For multi-labeled specimens it is easier to view the image in Split screen where each label is arranged side by side.
Page 74 OPERATION Quick start LSM 510 To make the image brighter or dimmer, adjust Detector Gain. This adjustment is very sensitive. Try using the left and right arrows to make the adjustment instead of dragging the slider bar. (10) To adjust the black level (background) use Ampl. Offset.
Page 75 OPERATION LSM 510 Quick start (12) Once you have optimized a particular channel, you can switch to the next channel desired and repeat steps 8, 9, and 10. (13) As soon as all channels are optimized, click on the <Stop> button.
Page 76 OPERATION Quick start LSM 510 (17) To further improve image quality you can slow the scan rate allowing more photons to integrate on the detector, or apply image averaging to remove random noise, or a combination of both. These adjustments are made by clicking on the <Mode> button on the Scan Control menu. Set the Scan Speed in the Speed area and Number in the Depth, Scan Direction &...
Page 77: Z Sectioning
OPERATION LSM 510 Quick start 4.3.7 Z Sectioning Once you have set up your image as defined in the above section, you can collect a series of confocal images through the different focal planes of your specimen. Click on the <Z Stack> button on the Scan Control menu.
Page 78 OPERATION Quick start LSM 510 Click on the <XY cont.> button to begin scanning. Fig. 4-20 Move the focus down manually (clockwise) until the image of the specimen begins to disappear, then click on the <Mark First> button in the Mark First/Last register.
Page 79 (11) A black bar will be shown under the image and will move from left to right, showing that the LSM 510 is in the process of Z sectioning. The laser will automatically stop scanning when z- sectioning is completed.
Page 80: Overview Of The Menu Items
Overview of Menu Items LSM 510 Overview of the Menu Items Start the LSM program as follows: Double-click on the Start LSM 510 icon on the desktop. Fig. 4-22 LSM 5 Switchboard menu The switchboard menu presents the following items for selection: Scan New Images Clicking on this button activates the complete LSM hardware.
Page 81 OPERATION LSM 510 Overview of Menu Items Operating modes Start Routine Mode Click on this button if you want to work with pre-configured system settings (typical applications). Start Expert Mode Use of this mode requires that you are thoroughly familiar with the exact microscope procedures and interrelations.
Page 82 OPERATION Overview of Menu Items LSM 510 Click on the <Start Expert Mode> button. The "LSM 510 - Expert Mode" main menu appears on the screen. Main menu Toolbar of File menu Fig. 4-23 The <File> button is active automatically, and the submenus selectable in it are shown in the second (bottom) toolbar.
Page 83 OPERATION LSM 510 Overview of Menu Items Process Used for the mathematical integration of acquired images. <Shift> allows you to perform a pixel shift correction. Toolbar of Process menu Fig. 4-25 For three-dimensional image processing. Toolbar of 3D View menu Fig.
Page 84 OPERATION Overview of Menu Items LSM 510 Options For custom-configuration of software and hardware options, and for exporting system operating sequences to the Routine Mode. Under this menu item access to the colouring table will be enabled. In the "Settings for User" window you can specify essential operating modes and informative help, organized by registers, which have an effect on the user interface.
Page 85: Image Acquisition (Acquire)
OPERATION LSM 510 Image Acquisition (Acquire) Image Acquisition (Acquire) In the main menu toolbar, click on <Acquire>. This opens another, subordinate toolbar in the main menu. Fig. 4-30 Toolbar subordinate to the <Acquire> item For preparing and acquiring a scanning image, it is recommended to call up and use the tools of the subordinate toolbar in succession from left to right.
Page 86: Laser Settings
OPERATION Image Acquisition (Acquire) LSM 510 4.5.1 Laser settings Click on the <Laser> button. This opens the "Laser Control" window, which shows all lasers connected to the system. The Laser Selection and Power Status panel, shows the types, operating statuses and excitation wavelengths of the lasers available.
Page 87 Switch on the Power Supply of the Enterprise laser, power potentiometer turned to maximum. Start the LSM 510 software. If the LSM 510 software is already running and you want to use the UV laser, do the following: Close the LSM 510 software.
Page 88: Microscope Settings (Conventional Microscopy)
OPERATION Image Acquisition (Acquire) LSM 510 4.5.2 Microscope settings (conventional microscopy) Place specimen on microscope stage. The cover slip must face up on an upright microscope, down on an inverted microscope. Select the objective to suit the job - dry, water or oil immersion (see inscriptions on objectives: w for water and oil for immersion oil).
Page 89 OPERATION LSM 510 Image Acquisition (Acquire) With Transmitted Light activated , the halogen lamp is automatically occluded in the laser scanning mode. Please bear in mind that the light intensity does not automatically correspond to 0 % when Light Remote is deactivated. The microscope setting (light intensity) of the last session, which was not remote-controlled, is restored on exit of the program.
Page 90 OPERATION Image Acquisition (Acquire) LSM 510 If you want to use an objective which is not contained in the nosepiece, proceed as follows: Change objectives Click on the "Change Objectives" icon. The submenu "Modify Objectives Settings" appears. Change Objectives icon Fig.
Page 91 OPERATION LSM 510 Image Acquisition (Acquire) Click on the <Maintain> button in the main menu and then on the <Reboot> button in the submenu. Confirm the selection in the "Reboot Components" dialog box by clicking on the <Reload Objectives from DB> button.
Page 92 OPERATION Image Acquisition (Acquire) LSM 510 4.5.2.1 Transmitted-light observation (Axioplan 2 Push in the silver slider (4-37/8) on the microscope tube as far as it will go. This opens the light path for specimen observation through the eyepieces. Actuate the shutter switch (4-37/4) to open the light path of the halogen lamp, and control its brightness with the potentiometer (4-37/3).
Page 93 OPERATION LSM 510 Image Acquisition (Acquire) 4.5.2.2 Epi-fluorescence observation (Axioplan 2 Turn on the HBO 100 W power supply with switch (4-37/2). Push in the silver slider (4-37/8) on the microscope tube as far as it will go. This opens the light path for specimen observation through the eyepieces.
Page 94 OPERATION Image Acquisition (Acquire) LSM 510 Other filter sets: The filter sets described in this section are included in the standard configuration, but other sets are available on request. DAPI: BP 365 FSET01 FT 395 LP 397 TRITC: BP 546...
Page 95 OPERATION LSM 510 Image Acquisition (Acquire) 4.5.2.3 Transmitted-light observation (Axiovert 100 M BP) Pull out the silver slider (4-39/1) on the microscope tube as far as it will go. This opens the light path for specimen observation through the eyepieces.
Page 96 OPERATION Image Acquisition (Acquire) LSM 510 4.5.2.4 Epi-fluorescence observation (Axiovert 100 M BP) Turn on the HBO 50 power supply switch (4-39/4). Pull out the silver slider (4-39/1) on the microscope tube as far as possible. This opens the light path for specimen observation through the eyepieces.
Page 97: Beam Path / Configuration
OPERATION LSM 510 Image Acquisition (Acquire) 4.5.3 Beam path / Configuration Click on the <Config> button. This opens the "Configuration Control" window, in which you can configure the system for scanning. The window contains two panels: Beam Path and Channel Assignment and Ratio Settings.
Page 98: Configuration
OPERATION Image Acquisition (Acquire) LSM 510 4.5.3.1 Taking over the configuration with Configuration Control After clicking on the <Recording Configurations> button, the Recording Configurations dialog box appears on the screen and allows the following settings to be made: Selection and calling up a configuration, whether factory-preset or created by the user on the Store /...
Page 99: Configuration
OPERATION LSM 510 Image Acquisition (Acquire) Click on the desired configuration. The selected configuration is shown in the Configurations status box. Click on the <Apply> button. This results in the stored instrument parameters being taken over for active use. The optical diagram of the configuration selected appears on the Beam Path and Channel Assignment panel.
Page 100: Configuration
This allows "cross-talking" from one channel to another being avoided when different tracks are used. Click on the <Acquire> and <Config> buttons from the LSM 510 tool bar. The Configuration Control menu appears on the screen.
Page 101: Configuration
OPERATION LSM 510 Image Acquisition (Acquire) Click on the <Show Tracks> button on the right side of the window. Within the "Configuration Control" window, the additional List of Tracks panel appears. The following functions are available in this dialog box: Fig.
Page 102: Configuration
OPERATION Image Acquisition (Acquire) LSM 510 <Add Track> button An additional track is added to the configuration list. The maximum of four tracks can be added. <Delete> button The track previously marked in the List of Tracks panel in the Name column is deleted.
Page 103 OPERATION LSM 510 Image Acquisition (Acquire) Fig. 4-45 <Frame> button The entire area is scanned, and then the parameters are reset. <Fast Switch> button The scanning procedure can be made faster. Only the Acousto-Optical Tunable Filters (AOTF) are switched, and no other hardware components.
Page 104: Configuration
OPERATION Image Acquisition (Acquire) LSM 510 Clicking the <Scan> button in the LSM 510 tool bar and the <Channels> button in the <Scan Control> window allows viewing of the channel configuration set before. Fig. 4-46 4-46 B 40-51 e 07/98...
Page 105 OPERATION LSM 510 Image Acquisition (Acquire) The following scan image shows the result with two defined tracks plus the ratio track and the overlay. Fig. 4-47 1. Track: Ratio-Track: Overlay 2. Track: -Ch 1-1 -R 1-1 -Ch 1-2 -Ch 3-1...
Page 106: Configuration
Clicking on the <Scan> button in the LSM 510 tool bar and the <Channels> button in the "Scan Control" window will display the defined channels and the ratio channel in the Channel Settings panel.
Page 107 OPERATION LSM 510 Image Acquisition (Acquire) Laser attenuation On the Beam Path and Channel Assignment panel, move the cursor to the <Excitation> button. Once the cursor has changed into a hand symbol, click on the button. This opens a drop-down list of all available lasers with their wavelengths and their usable AOTF attenuation.
Page 108 OPERATION Image Acquisition (Acquire) LSM 510 Use the AOTF to set the laser intensity not completely to zero, but only to 0.1 %. This allows you to adapt the laser intensity very sensitively to the job. Click on <Line On>.
Page 109 OPERATION LSM 510 Image Acquisition (Acquire) Beam path - Main beam splitter On the Beam Path and Channel Assignment panel, move the cursor to the symbol of the main beam splitter HFT Click on the symbol once the cursor has changed into a hand.
Page 110 OPERATION Image Acquisition (Acquire) LSM 510 Clicking on the OFF spot results in deactivation of the corresponding channel. Further colors for the corresponding channel can be produced as follows: Clicking on the <Define> button will open a further "Channel Colors" window. Via a reticule in the "Define Color"...
Page 111: Configuration
OPERATION LSM 510 Image Acquisition (Acquire) Emission filter On the Beam Path and Channel Assignment panel, move the cursor to the emission filter symbol. Click on the symbol once the cursor has changed into a hand. This opens a drop-down list of all available emission filters (e.g.
Page 112: Scanning Modes
OPERATION Image Acquisition (Acquire) LSM 510 4.5.4 Scanning modes Taking a simple configuration as an example, the sections below describe the procedure for acquiring a single scanned image in single-channel presentation, using fluorescence and confocal imaging. Requirements Axioplan 2 Push in the silver slider (4-37/8) on the microscope tube as far as it will go.
Page 113 OPERATION LSM 510 Image Acquisition (Acquire) 4.5.4.1 Frame Before starting, click on the <Mode> button on the upper toolbar. Click on <Frame> on the lower toolbar. The toolbar changes to correspond to the Frame selection. On the Objective Lens & Image Size panel,...
Page 114 OPERATION Image Acquisition (Acquire) LSM 510 On the Zoom, Rotation & Offset panel, use the slider to set the desired zoom factor between 1 and 8. This adjustment is infinite. The scanning frame thus defined is shown by a red outline.
Page 115 OPERATION LSM 510 Image Acquisition (Acquire) 4.5.4.2 Z mode This mode allows you to create a stack of images at different Z levels (slices). In the "Scan Control" window on the <Z Stack> button. This opens the Z Settings panel in the "Scan Control"...
Page 116 OPERATION Image Acquisition (Acquire) LSM 510 The Optical Slice dialog box displays the following information: Black: Stack Z Size (µm) intervals x (number of slices - 1) Optimal Interval depending on the objective used and the pinhole diameter setting Red and other colors: Presentation of the actual data set by the operator helps to optimize stack creation.
Page 117 OPERATION LSM 510 Image Acquisition (Acquire) Cutline and Range For a useful evaluation of the z stack you must proceed as follows: Click on the <Cutline> button. The image window shows the course of the red cutline. Clicking on the cutline allows you to remove the line to the position where the specimen should be cut.
Page 118 OPERATION Image Acquisition (Acquire) LSM 510 Click on the <Range> button. The image window marks the range to be scanned by red outlines. Not only the current slice (green cutline) but also the red range lines can be removed by moving the cursor in the image window.
Page 119 OPERATION LSM 510 Image Acquisition (Acquire) Mark First/Last Optimum stack size can be obtained by manual focusing during scan movement to the level where the stack should start. Proceed as follows Open the Mark First/Last register. Start the scanning process by clicking the <XYcont>...
Page 120 OPERATION Image Acquisition (Acquire) LSM 510 When the <Z Stack> and <Mode> buttons are activated, objective can be changed, number of pixel and scan speed can be set; desired average and number of gray shade can be chosen as well as uni- or bidirectional scan;...
Page 121 A scan image allows certain areas (ROIs) to be defined. Only these areas of interest will be scanned. The laser beam will be switched in only in these areas via AOTF. Click on the <Acquire> and <Edit ROI> buttons from the LSM 510 tool bar. The "Edit ROI" window appears on the screen.
Page 122: Configuration
OPERATION Image Acquisition (Acquire) LSM 510 The "Add ROI List" window will appear. Enter any required name to store the ROIs and click on the <OK> button. Fig. 4-65 This stored ROI configuration appears in the ROI Lists panel of the "Edit ROI" window.
Page 123 OPERATION LSM 510 Image Acquisition (Acquire) The following is an example of a scanning procedure where the ROI function was activated. Only the regions of interest defined before are visible in the scanning image, the other areas remain dark. Fig. 4-66...
Page 124 Click on the <Acquire> and <TimeSeries> buttons in the LSM 510 tool bar. The "Time Series Control" window appears on the screen. Set the relevant parameters for time control in the Start Series, End Series and Cycle Delay boxes.
Page 125 OPERATION LSM 510 Image Acquisition (Acquire) "Time Series Control" window enabling the entry of parameters for time series control. Fig. 4-67 B 40-051 e 07/98 4-67...
Page 126 OPERATION Image Acquisition (Acquire) LSM 510 The following example of a scanning image was taken using the Time Series function. Both the time and the markers set during the scanning procedure are projected in the image series in different colors.
Page 127 OPERATION LSM 510 Image Acquisition (Acquire) 4.5.4.5 Edit Bleach function The use of this function permits the intense bleaching of a defined sample area. To use the Edit Bleach function, proceed as follows: Click on the <Edit Bleach> button. The "Bleach Control" window appears.
Page 128 OPERATION Image Acquisition (Acquire) LSM 510 Click on the <Define Region> button. The "Bleach Regions" window appears. Define the required bleach regions in the scan image. Select the required laser wavelength and its intensity under Excitation of Bleach Track in the "Bleach Control"...
Page 129: Routine Mode
Standard Examination Methods or by User Defined Examination Methods. Standard Examination Methods are included in the LSM 510 software package and must only be activated once during the first application of the routine mode. User Defined Examination Methods...
Page 130 Click on the <Close> button in the "Select Method To Add" window. The standard examination methods are taken over and are then available in the LSM 510 switchboard menu. The relevant labeling appears beside the buttons. Fig. 4-73 Fig. 4-74...
Page 131: Apply Standard Examination Methods In The Routine Mode
OPERATION LSM 510 Routine Mode 4.6.2 Apply Standard Examination Methods in the Routine Mode Click on the button of the required standard examination method, e.g. Standard: Rhodamine / FITC. The "Routine Mode - Microscope Setup" window will appear. Prepare your specimen for examination in the same way as in the Expert Mode.
Page 132 OPERATION Routine Mode LSM 510 Click on <Save> or <Save As> to store the acquired image. Click on <Finish> to exit the Routine Mode and return to the LSM 510 switchboard menu. Fig. 4-77 4-74 B 40-051 e 07/98...
Page 133: Export User-Defined Examination Methods To The Routine Mode
Routine Mode 4.6.3 Export User-Defined Examination Methods to the Routine Mode Click on the <Start Expert Mode> button (Fig. 4-71) in the LSM 510 switchboard menu. The "LSM 510 - Expert Mode" main menu appears on the screen. Fig. 4-78 Load a Stack or Time Series image which was scanned using optimized parameters.
Page 134: Activate User-Defined Examination Methods In The Routine Mode
Routine Mode. Fig. 4-80 Exit the Expert Mode by clicking on <File> and then on <Exit>. Fig. 4-81 The LSM 510 switchboard menu appears on the screen again. 4.6.4 Activate User-Defined Examination Methods in the Routine Mode Start the Routine Mode.
Page 135: Apply User-Defined Examination Methods In The Routine Mode
OPERATION LSM 510 Routine Mode Click on the <Standard> or the <Mark for List> button. Close the "Select Method To Add" window by clicking on <Close>. The "Routine Mode - Select Examination Method" window appears on the screen again. Depending...
Page 136: Acquisition Of A Z Stack In The Routine Mode
OPERATION Routine Mode LSM 510 4.6.6 Acquisition of a Z Stack in the Routine Mode Load the required stack, the parameters of which you want to use for further work. The "Routine Mode - Microscope Setup" window becomes visible. Prepare your specimen for examination in the same way as in the Expert Mode.
Page 137 OPERATION LSM 510 Routine Mode If you are satisfied with the setting, start by clicking on <Next>. The "Routine Mode - Image Acquisition" window appears (Fig. 86). Click on <Start> to trigger acquisition of the stack. Fig. 86 If you want to correct the parameters offered, click on the <Make Z Cut>...
Page 138 OPERATION Routine Mode LSM 510 You can now select Z-stack limits (symmetrically to the central Z-frame), the number of steps and the interval size. Fig. 88 Further modifications of the stack parameters can be performed as follows: Click on the <Use New Parameter> button.
Page 139: Image Optimization
OPERATION LSM 510 Image Optimization Image optimization Single frames Described below is the example of the acquisition of an image, using an excitation wavelength of 488 nm and a fluorescence emission range above 505 nm. Let the specimen be a thin section through a stem of Convallaria majalis (Lily-of-the-Valley). The description applies to the use of the Axioplan microscope, and analogously also to the Axiovert.
Page 140 OPERATION Image Optimization LSM 510 In the main menu click on the <Scan> button. This opens the "Scan Control" window. Click on the <Mode > button. For a frame scan, click on the <Frame> button. On the Objective Lens & Image Size panel, select Objective and Frame size for the scan (e.g.
Page 141 OPERATION LSM 510 Image Optimization Click on the <Channels> button. This displays the preset parameters of the configuration loaded. Fig. 4-92 B 40-051 e 07/98 4-83...
Page 142 OPERATION Image Optimization LSM 510 Click on the <Find> button. Make sure to position the slider correctly. Then scan while the slider is in the LSM position. This starts the scanning process. The image is seen to build up gradually in a new window.
Page 143: Detector Gain/Ampl. Offset/Ampl. Gain
OPERATION LSM 510 Image Optimization 4.7.1 Detector Gain/Ampl. Offset/Ampl. Gain In the "Scan Control" window, click on the <Cont.> button. This starts a continuous scan. Click on the <Palette> button of the image processing toolbar. This opens the "Color Palette" window.
Page 144 OPERATION Image Optimization LSM 510 In the Color Palette List panel, click on the "Range Indicator" item. The scanned image appears in a false-color presentation. If the image is too bright, it appears red on the screen. Fig. 4-95 4-86...
Page 145 OPERATION LSM 510 Image Optimization If the image is not bright enough, it appears blue on the screen. Fig. 4-96 B 40-051 e 07/98 4-87...
Page 146 OPERATION Image Optimization LSM 510 On the Channel Settings panel of the "Scan Control" window, set the PMT gain with the Detector Gain slider. The image should not have more than a trace of red. Adjust the Ampl. Offset slider so that areas without picture content just show a trace of blue.
Page 147: Pinhole Adjustment
OPERATION LSM 510 Image Optimization 4.7.2 Pinhole adjustment In all existing standard configurations, the pinholes have already been adjusted at the factory. These settings are taken over for active operation when a standard configuration is loaded. If you want to create a setting that differs from the standard configurations, adjust the pinhole...
Page 148 OPERATION Image Optimization LSM 510 Click on the <Pinhole> button. This opens the "Pinhole & Collimator Control" window. Select the pinhole to be adjusted from the Description list box. If several channels are used to produce the image, all the used pinholes must be adjusted separately.
Page 149 In all of the available standard configurations, the pinhole positions are factory-adjusted and are activated when a standard configuration is loaded. The automatic adjustment allows the LSM 510 pinholes to be used with any combination of beam splitters. Click on the < Adjust Automatically > button.
Page 150 OPERATION Image Optimization LSM 510 Click on the <Optimal Position> button in the Collimator box. Optimum positioning of the collimator will be performed. The <Default Position> button enables the collimator to be set back to the factory-adjustment. Note: A change of the pinhole diameter made manually in the Pinhole box will not be activated in the Scan Control box.
Page 151: Scan Speed
OPERATION LSM 510 Image Optimization 4.7.3 Scan speed The signal-to-noise ratio can be substantially improved by reducing the scanning speed to an acceptable level and averaging over several scans (i.e. with an Average Number greater than 1 in the "Scan Control" window).
Page 152 OPERATION Image Optimization LSM 510 Multiple-channel Requirements: The suitable lasers are on. The specimen has been positioned and focused for scanning. The slide rod on the microscope tube has been pulled out as far as it will go. In the "Acquire" main menu item, click on the <Config>...
Page 153 OPERATION LSM 510 Image Optimization Click on the <Scan> button in the main menu. This opens the "Scan Control" window. In the "Scan Control" window, set the parameters in the same way as described for single-channel presentation. Click on the <Find> button in the "Scan Control" window.
Page 154 OPERATION Image Optimization LSM 510 Click on the <Channels> button in the "Scan Control" window. This opens the Channel Settings and Excitation of Track panels. The channels used are color-highlighted. The image optimization processes Detector Gain / Ampl. Offset / Ampl. Gain...
Page 155 OPERATION LSM 510 Image Optimization Click on the <Display - Split xy> button of the image processing toolbar. This displays the separate images scanned in the channels and the composite image. Scan image 1 Scan image 2 Cumulative scan image Fig.
Page 156: Channel Shift Function
The Channel Shift function is used to produce a congruent image with relation to the pixels of the various channels. This pixel correction function is particularly important in UV applications. Click on the <Process> and <Shift> buttons from the LSM 510 tool bar. The Channel Shift menu appears on the screen. Fig. 4-106 Select the channels required for processing in the Shift box by clicking on the <Ch1>...
Page 157 OPERATION LSM 510 Image Optimization The following image shows the result of a pixel shift via the Shift function. This image change can be stored in the image database via the <Save> or <Save As> buttons. Fig. 4-107 For applications requiring 3- or 4-channel scanning, proceed in the same way as described for the 1- or 2-channel mode.
Page 158 OPERATION Image Optimization LSM 510 For overlaying fluorescence and transmitted-light images, click on the <Transmission> button on the Beam Path and Channel Assignment panel. The transmitted light PMT will be switched active. Of course, all other transmitted light applications like...
Page 159: Analysis Of Images And Stacks
OPERATION LSM 510 Analysis of Images and Stacks Analysis of Images and Stacks Call up an image just scanned, or an image from the database. Fig. 4-109 The image information called up is shown in a window. The two columns of buttons headed "Select"...
Page 160 OPERATION Analysis of Images and Stacks LSM 510 (1) Select - Chan You can assign any color from the color palette to a channel, so as to highlight or suppress certain image details. Fig. 4-110 (2) Select - Zoom The image can be zoomed by various methods.
Page 161 OPERATION LSM 510 Analysis of Images and Stacks Zoom-+ Enlarges the image by factor 2. Zoom-– Reduces the image by factor 2. Zoom 1:1 Restores an image zoomed in any way to its original size. Zoom-Mouse Allows you to enlarge/reduce an image using the left/right mouse button, provided that the cursor is inside the image.
Page 162 OPERATION Analysis of Images and Stacks LSM 510 (3) Select - Slice This function allows you to select and view individual slices from a stack. Fig. 4-112 Example: Slice No. 4 from a stack of 15 slices Select the slices using the slider on the right.
Page 163 OPERATION LSM 510 Analysis of Images and Stacks (4) Select - Overlay This option makes available a number of measurement functions, such as length, angle, area and circumference measurements. Furthermore, you can enter comments into the image. Measurement functions Text...
Page 164 OPERATION Analysis of Images and Stacks LSM 510 (5) Select - Contr This function allows you to vary the contrast and brightness of an image, either separately for each channel or jointly for all channels. Further image processing possibilities can be activated or deactivated alternately using the <More>...
Page 165 OPERATION LSM 510 Analysis of Images and Stacks (7) Select - Anim This option allows you to call up the individual slice images of a stack in continuous succession (animation). The additional Animate dialog box allows you to influence the animation action.
Page 166 OPERATION Analysis of Images and Stacks LSM 510 (9) Select - Crop Here, you can define an area of any size, position and rotation which you want to scan. Offset: Click into the square (scanning field), keep the left mouse button pressed and drag the square to the required position.
Page 167 OPERATION LSM 510 Analysis of Images and Stacks (10) Select - Copy Copies the image content to the clipboard, from where the image can be accessed and copied to other programs/applications (e.g. the MS-WORD word processor). (11) Select - Save Allows you to save the scanned image(s) under the existing name.
Page 168 OPERATION Analysis of Images and Stacks LSM 510 (13) Display - xy Allows you to switch back to the original image from a different presentation mode, such as Split-Mode or Gallery. (14) Display - Split xy In multiple-channel scanning, the image is seen to be generated in the different channels, together with the composite image.
Page 169 OPERATION LSM 510 Analysis of Images and Stacks (15) Display - Ortho Orthogonal sections can be made anywhere in a stack. Furthermore, it is possible to measure spatial distances. Clicking on "Display-Ortho" makes section lines and the correlated section projections in the image appear.
Page 170 OPERATION Analysis of Images and Stacks LSM 510 By changing the parameters X, Y and Z on the Orthogonal Sections panel, the section plane can be positioned at liberty within the specimen. The position of the section plane is shown by colored lines.
Page 171 OPERATION LSM 510 Analysis of Images and Stacks You can reposition the (red) YZ plane in the same way using the symbol. The result of an orthogonal section is visible at the image margin, no matter which method you used.
Page 172 OPERATION Analysis of Images and Stacks LSM 510 (16) Display - Cut In a stack you can generate sections along a plane of freely selectable positions. Clicking on the <Display-Cut> button opens the Cut dialog panel to the right of the image processing toolbar.
Page 173 OPERATION LSM 510 Analysis of Images and Stacks (17) Display - Gallery The various slices of a stack are shown in a tiled presentation of several rows (called a gallery), in chronological order of their generation. A click on the <Display-Gallery> button not only produces the gallery itself but also the Gallery panel with two buttons: <Data>...
Page 174 OPERATION Analysis of Images and Stacks LSM 510 Clicking on the <Data> button causes the Z data to be entered into the images of the stack. This provides you with information on the Z distance of each section (slice) relative to the first section plane.
Page 175 OPERATION LSM 510 Analysis of Images and Stacks (18) Display - 2.5D Clicking on this button displays an image in a pseudo-3D mode, representing the intensity distribution over the scanned area, and opens a panel headed "Pseudo 3D", in which you can select various presentations with the <Profile>, <Grid>...
Page 176 OPERATION Analysis of Images and Stacks LSM 510 (19) Display - Histo The histogram function displays a graph of the intensity distribution of an image as well as information on the frequency of the various intensities, separate for each channel.
Page 177 OPERATION LSM 510 Analysis of Images and Stacks (20) Display - Profile The Display-Profile function shows the intensity profile across the image along a freely selectable line. In multiple-channel mode, the intensity profile is shown separately for each channel. The intensity curves are shown in a graph below the scanned image.
Page 178 OPERATION Analysis of Images and Stacks LSM 510 You can place markings wherever you like and follow up this line detecting the intensity profile. Freely select- able line for intensity profile Marking Fig. 4-128 Click on the <Diagr. in Image> button to overlay an intensity graph directly on the image.
Page 179 OPERATION LSM 510 Analysis of Images and Stacks (21) Display - Coloc. The Display-Coloc. function presents a comparison between two images by computing a scatter diagram (colocalization). 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 brightness level of pixel P1 is interpreted as X coordinate, and that of pixel P2 as Y coordinate of the scatter diagram.
Page 180 OPERATION Analysis of Images and Stacks LSM 510 Scatter diagram Colocalization dialog box Fig. 4-130 Identical images produce a clean diagonal line running from bottom left to top right, because only pixel pairs (0,0), (1,1), (2,2) etc. can occur. Differences between the images cause irregular spots in the scatter diagram.
Page 181 OPERATION LSM 510 Analysis of Images and Stacks (22) Display - Area Clicking on the <Display-Area> button opens the Area Measure dialog box. The top area of this panel shows the geometric size of the scanned image. This function allows you to measure the area of any plane geometric figure within the scan image. The function can be activated by clicking on one of the geometry buttons, e.g.
Page 182 OPERATION Analysis of Images and Stacks LSM 510 Clicking on the <Flood fill> button (paint jar) and moving the cursor to the area to be excluded causes the remaining area to be computed and the result indicated under Area Measure.
Page 183 OPERATION LSM 510 Analysis of Images and Stacks If you specify a top and bottom intensity threshold, the area lying within this intensity interval can be computed. Specify the thresholds either with the Threshold low and Threshold high sliders, or with the buttons.
Page 184 OPERATION Analysis of Images and Stacks LSM 510 (23) Display - Prev. This function enables you to assemble a preview of all pictorial, textual and graph information you want to print out. The size and position of the image can be varied using the mouse pointer in the image window.
Page 185 OPERATION LSM 510 Analysis of Images and Stacks Assembly of image, intensity profile and scan info Fig. 4-134 B 40-051 e 07/98 4-127...
Page 186 OPERATION Analysis of Images and Stacks LSM 510 A layout generated with Prev. (Preview) can be printed by clicking on the <Print-Print> button. Clicking on the <Print-Setup> button opens the "Print Setup" window, in which you can specify print parameters.
Page 187 OPERATION LSM 510 Analysis of Images and Stacks (24) Display - Info Clicking on the <Info> button, all parameters used to generate the image appear at the left image side. Image parameters Fig. 4-136 B 40-051 e 07/98 4-129...
Page 188 OPERATION Analysis of Images and Stacks LSM 510 (25) 3D View For the further three-dimensional analysis of image sequences (stacks), click on the <3D View> button in the main menu. This opens a submenu bar with the buttons <Depth Cod.>, <Projection> and <Stereo>.
Page 189 OPERATION LSM 510 Analysis of Images and Stacks The "Preview" function permits you to regard the influence of parameter changes in an image window. After finding the optimum adjustment using the "preview" function, you have to generate the final version of the image using the <Apply> button, and the print will be prepared.
Page 190 OPERATION Analysis of Images and Stacks LSM 510 (b) Projection Requirement: A stack of images must be available. Clicking of the <Projection> button opens the "Projection" window. On the Projection panel, set the parameters needed for the animation: Turning Axis, First Angle, Number Projection and Difference Angle.
Page 191 OPERATION LSM 510 Analysis of Images and Stacks The computed 3D sequence can be animated with the <Anim> button in the Select bar. In addition, the "Animate" dialog box appears, in which you can influence the direction and speed of 3D image rotation.
Page 192 OPERATION Analysis of Images and Stacks LSM 510 To view the computed 3D sequence as a gallery on the screen, click on the <Display-Gallery> button. Fig. 4-142 4-134 B 40-051 e 07/98...
Page 193 OPERATION LSM 510 Analysis of Images and Stacks (c) Stereo Requirement: A stack of images must be available. Clicking on the <Stereo> button opens the "Stereo Images" window. The image to be processed appears on the Source panel. On the Stereo Images panel, set the parameters needed for stereoscopic viewing, such as...
Page 194 OPERATION Analysis of Images and Stacks LSM 510 To start computation of the stereoscopic image, click on the <Apply> button. The image is built up twice (once each for the red and green colors), resulting in a stereoscopic image. The stereoscopic effect can only be seen with the aid of red/green 3D goggles.
Page 195 OPERATION LSM 510 Analysis of Images and Stacks On clicking on the <Apply> button, the two stereo mates are presented side by side and can be viewed without red/green 3D goggles. Fig. 4-145 B 40-051 e 07/98 4-137...
Page 196: Data Base / Loading And Storing Of Images
OPERATION Database / Loading and Storing of Images LSM 510 Data base / Loading and Storing of images All the generated images are stored in existing or new databases (*.mdb). To load an image, the relevant database must be opened first. Individual images can be loaded using the Import function.
Page 197 OPERATION LSM 510 Database / Loading and Storing of Images Enter the name of the database you want to create in the File name text box, e. g. "Convallaria". If you want to create the image database in a certain folder (drive/directory), click on the arrow button next to the Create in box.
Page 198 OPERATION Database / Loading and Storing of Images LSM 510 Click on the <Create> button. This creates the new image database in the selected drive and directory. The "Convallaria.mdb" window appears, presenting the opened database with 0 of 0 image entries.
Page 199: Loading An Image From Database
OPERATION LSM 510 Database / Loading and Storing of Images 4.9.2 Loading an image from database Click on the <File> button of the main menu toolbar. This opens another, subordinate toolbar in the main menu. Fig. 4-149 Subordinate toolbar for File menu item Click on the <Open>...
Page 200 Database / Loading and Storing of Images LSM 510 This opens a window, e.g. Multi Channel 2_0.mdb - LSM 510, with buttons which can be used to call up the individual images in the database and to have them presented in various ways.
Page 201 OPERATION LSM 510 Database / Loading and Storing of Images Click on the <Gallery> button. All images of the image database, e.g. Multi Channel 2_0.mdb, (image series) are shown in a tiled arrangement on the screen. Fig. 4-153 To select one of the images of the series for normal-size presentation, double-click on the desired image.
Page 202 OPERATION Database / Loading and Storing of Images LSM 510 Image processing and analysis can be effected via the two-column toolbar (see section 4.8) Fig. 4-154 4-144 B 40-051 e 07/98...
Page 203: Saving An Image
OPERATION LSM 510 Database / Loading and Storing of Images 4.9.3 Saving an image Click on the <File> button of the main menu toolbar. This opens another, subordinate toolbar in the main menu. Click on the <Save> or <Save As> button.
Page 204 OPERATION Database / Loading and Storing of Images LSM 510 Enter the name of the image in the Name textbox, e.g. Spores. Click on the <New MDB> button. This opens the "Create New Database" window in which you can create a new image database.
Page 205 OPERATION LSM 510 Database / Loading and Storing of Images The "Single Channel 2_0.mdb-LSM510" window appears. The window now shows the saved image. The Recordset box indicates the current number of the image in the image series contained in this database.
Page 206: Import Of Images
OPERATION Database / Loading and Storing of Images LSM 510 4.9.4 Import of images Click on the <File> button of the main menu toolbar. This opens another, subordinate toolbar in the main menu. Fig. 4-159 Subordinate toolbar for the File menu item Fig.
Page 207: Export Of Images
OPERATION LSM 510 Database / Loading and Storing of Images 4.9.5 Export of images The <Export> function allows the export of both newly scanned images and images from the database. For this, the relevant image must be created or loaded.
Page 208 OPERATION Database / Loading and Storing of Images LSM 510 Notes: System backup A complete backup is contained on the enclosed optical disk. User files backup The following user-generated files need to be included in a backup procedure (keep directory structure): Image database files *.mdb (but not system_configuration_*.mdb...
Page 209: Macro
Macro language in the "Microsoft Word for Windows" and "Microsoft Excel for Windows" products. Experience with "Microsoft Visual Basic" would also be beneficial for macroprogramming of the LSM 510. An Integrated Development Environment, called IDE in the following, is available for the editing and debugging of macros.
Page 210 OPERATION Macro LSM 510 Fig. 4-164 This window allows you to manage project data and to allocate macros to the buttons in the main window. Before you can record or edit a macro, you have to create a project as follows: Press the <New>...
Page 211: Recording And Running Of Macros
OPERATION LSM 510 Macro 4.10.3 Recording and running of macros Before recording a command sequence, you can enter the name for the macro to be created in the Rec Name editing box of the "Recording" dialog box. The following buttons are used for recording and running: "Recording"...
Page 212: Assignment Of Macros To The Macro Buttons In The Main Window
OPERATION Macro LSM 510 4.10.4 Assignment of macros to the macro buttons in the main window Press the <Assign Macro to Button> button to switch to the "Define Buttons" dialog box. Fig. 4-165 Select the button number from the "Button" box Select the button labelling in the "Text"...
Page 213: Editing And Debugging Of Macros
OPERATION LSM 510 Macro 4.10.5 Editing and debugging of macros The <Edit> button activates IDE which allows macros to be edited and debugged. Under the "Help - Contents and Index" menu item, IDE contains detailed "online" help on its operation and on the VBA macro language.
Page 214: Shut-Down Procedure
This closes the respective window and removes the respective icons from the taskbar. After all dialog windows have been closed, the "LSM 510 Switchboard" window appears. Fig. 4-166 Click on the <Exit> button.
Page 215: Running Down The Operating System
OPERATION LSM 510 Shut-Down Procedure 4.11.2 Running down the operating system Move the cursor to the bottom margin of the screen. This opens the taskbar containing the <Start> button. Click on the <Start> button of the taskbar. This opens a pop-up menu.
Page 216 OPERATION Shut-Down Procedure LSM 510 This opens the "Shut Down Windows" window, in which you can select between Shut down, Restart and Login. Fig. 4-168 Unless already set by default, click on "Shut down the computer?". Click on the <Yes> button.
Page 217 Fig. 4-169 (3) Turning power off Throw the REMOTE CONTROL main switch to position "OFF". This puts your LSM 510 microscope system, including the computer, off power. B 40-051 e 07/98 4-159...
Page 218: Annex
OPERATION Annex LSM 510 4.12 Annex 4.12.1 Application-specific configurations Application-specific configurations for module 510-1 (458/488, 543), 2 PMTs Lucifer Yellow FITC (Cy2) Rhod (Cy3, TexRed) Laser NFT 1 none none none NFT 2 NFT 3 none none none EM 1...
Page 219 OPERATION LSM 510 Annex Application-specific configurations for module 510-2 (488/568), 2 PMTs FITC (Cy2) Rhod (Cy3, TexRed) FITC/Rhod Laser 488, 543 488/568 none none NT 570 NFT 1 NFT 2 mirror none none plate NFT 3 EM 1 LP 505...
Page 220 OPERATION Annex LSM 510 Application-specific configurations for module 510-3 (458/488, 543, 633), 3 PMTs Lucifer Yellow FITC (Cy2) Rhod (Cy3, TexRed) Laser NFT 1 none none none none NFT 2 NFT 3 none none none none EM 1 LP 475...
Page 221 OPERATION LSM 510 Annex Application-specific configurations for module 510-4 (488/568, 633), 3 PMTs FITC (Cy2) Rhod (Cy3, TexRed) Laser NFT 1 none none none NFT 2 NFT 3 none none none EM 1 LP 505 LP 585 LP 650 EM 2...
Page 222 OPERATION Annex LSM 510 Application-specific configurations for module 510-5 (351/364, 458/488, 543), 3 PMTs DAPI Lucifer Yellow FITC (Cy2) Rhod (Cy3, TexRed) Laser UV (375) NFT 1 none none none none NFT 2 NFT 3 none none none none EM 1...
Page 223 OPERATION LSM 510 Annex Application-specific configurations for module 510-6 (351/364, 458/488, 543, 633), 4 PMTs DAPI Lucifer Yellow FITC (Cy2) Rhod (Cy3, TexRed) Laser UV (375) UV/488/543/633 NFT 1 none none none none none NFT 2 NFT 3 none none...
Page 224 OPERATION Annex LSM 510 DAPI/FITC/Rhod/ DAPI (nb) FITC (nb) Rhodamine (nb) Laser 364, 488, 543, UV/488/543/633 UV (375) NFT 1 NT 545 mirror mirror mirror NFT 2 NT 490 mirror plate plate NFT 3 NT 635 Vis EM 1 LP 650...
Page 225 OPERATION LSM 510 Annex Application-specific configurations for module 510-7 (351/364, 488/568, 633), 4 PMTs DAPI FITC (Cy2) Rhod DAPI/FITC (Cy3, TexRed) Laser 364, 488 UV (375) UV/488/568/633 UV/488 NFT 1 none none none none NT 490 NFT 2 mirror NFT 3...
Page 226 OPERATION Annex LSM 510 DAPI/FITC/Rhod DAPI/FITC/Rhod/ DAPI/FITC/Cy5 DAPI/Rhod/Cy5 FITC/Rhod/Cy5 Laser 364, 488, 568 364, 488, 568, 633 364, 488, 633 364, 568, 633 488, 568, 633 UV/488/568/633 UV/488/568/633 UV/488/568/633 UV/488/568/633 UV/488/568/633 NFT 1 NT 570 NT 570 NT 570 NT 570...
Page 227 OPERATION LSM 510 Annex Application-specific configurations for module 510-8 (458/488, 543, 633), 2 PMTs Lucifer Yellow FITC (Cy2) Rhod (Cy3) Laser NFT 1 none none none none NFT 2 NFT 3 none none none none EM 1 LP 475 LP 505...
Page 228 OPERATION Annex LSM 510 Application-specific configurations for module 510-9 (351/364, 458/488, 543), 2 PMTs DAPI Lucifer Yellow FITC (Cy2) Rhod (Cy3) Laser 351/364 UV (375) NFT 1 none none none none NFT 2 NFT 3 none none none none EM 1...
Page 229: Filter Change In The Detection Beam Path Of Channels 1 And 2
OPERATION LSM 510 Annex 4.12.2 Filter change in the detection beam path of channels 1 and 2 For optimum investigation of specimens it is useful to employ filter wheels permitting motor-controlled change between different filters for narrow-band or broad-band detection depending on the wave- length.
Page 230: Detaching / Attaching The Scanning Module From / To Microscope Stands
457411-9011 (CAN-BUS) and 457411-9012 (RS 232) from the Axioplan, plug them into the Axiovert and secure them there. Switch the LSM 510 on with the REMOTE switch. Click on the "Axiovert.ico" icon to update the system database with the new database of the Axiovert 100 M microscope.
Page 231 OPERATION LSM 510 Annex Fig. 4-171 Change-over of the Scanning Module B 40-051 e 07/98 4-173...
Page 232: Hints On The Use Of The Hrz 200 Fine Focusing Stage
OPERATION Annex LSM 510 4.12.4 Hints on the use of the HRZ 200 fine focusing stage 4.12.4.1 General description The HRZ 200 fine focusing stage is a compact attachment for the Axioplan 2 and Axiovert 100 M microscope stages, which allows the particularly fast and high-precision fine focusing of the object. The HRZ 200 permits fine focusing over a range of 200 µm, with the smallest step width being less than...
Page 233 4.12.4.3 Operation Fine focusing Select the <Acquire> button from the LSM 510 main menu. Select the <Stage> button. The following menu appears: Fig. 4-172 The <Hrz Step> slider is used to set the step width of the fine focusing stage.
Page 234 OPERATION Annex LSM 510 Z Mode via the HRZ 200 This mode is used to acquire two- or three-dimensional stacks of images in different z-positions, depending on whether <Line> or <Frame> scan has been activated. The benefit of this mode over <Z Sectioning>...
Page 235 4.12.4.4 Additional information on the operation The HRZ 200 fine-focusing stage is a high-precision, sensitive accessory for the LSM 510 from Carl Zeiss and must therefore be treated carefully. High mechanical stress, such as the use of specimens weighing more than 100 g or the application of pressure or knocks on the movable stage tongue, can result in damage and therefore in failure of the stage function.
Page 236 OPERATION Annex LSM 510 The shift is read off from the microscope stages. In the case of the manual Axioplan 2 stage, x can be read directly from the scale adhered to the front of the stage. In the case of the manual Axiovert 100...
Page 237: Scanning Stages
OPERATION LSM 510 Annex 4.12.5 Scanning stages The following software description applies to systems which are equipped with a scanning stage. 4.12.5.1 Routine Mode In the Routine Mode, the Standard Examination Methods panel enables you to reach the "Routine Mode - Microscope Setup" window Clicking on the <Stage>...
Page 238 OPERATION Annex LSM 510 XY Step Size The required step size for the scanning stage can be set in three ways: 1.) by shifting the slider 2.) by clicking on the arrow keys; clicking on the right arrow key increases the step width, clicking on the left arrow key decreases the step width.
Page 239 OPERATION LSM 510 Annex <Z> Moves Clicking on the Up arrow key moves the specimen stage/nosepiece upwards (the distance between objective and specimen is reduced). Clicking on the Down arrow keys moves the specimen stage/nosepiece downwards (the distance between objective and specimen is increased).
Page 240 OPERATION Annex LSM 510 4.12.5.2 Expert Mode The following window opens in the Expert Mode after clicking on the Stage button in <Acquire>: Fig. 4-175 This menu enables control of the scanning stage; a session-related MARK & FIND function is available to you.
Page 241 OPERATION LSM 510 Annex The Current Position and Selected Position display for X and Y is below. Below that, you will find the table of marked positions and the possibility to activate and delete them. Moving the scanning stage The scanning stage can be moved using the joystick, or software-controlled using the sliders.
Page 242 OPERATION Annex LSM 510 <Zero> Zeros the Current Position display and thus sets the currently set stage position to 0 in relation to X and Y. The already marked object areas thus receive new X and Y-coordinates. <Current Position> Current Position displays the currently set stage position in relation to the zero position.
Page 243: Specification Of Trigger-Interface Lsm510
OPERATION LSM 510 Annex 4.12.6 Specification of Trigger-Interface LSM510 Application: With the LSM510 Release 2.01 you can control various actions externally using Trigger-In or force external devices to work at a defined time depending on an action using Trigger-Out during time series.
Page 244 OPERATION Annex LSM 510 Trigger pulse description: Level detection: Low level: 0.0 - 1.0 V High level: 3.0 - 5.0 V Slew rate: rising edge: 1 µs falling edge: 1 µs Pulse width (always positive pulses/high level): Trigger-In: 20 ms...
Page 245 OPERATION LSM 510 Annex Pin assignment: Name Direction Description Trig1O Trigger Output #1 Trig2O Trigger Output #2 Trig3O Trigger Output #3 Trig4O Trigger Output #4 5...8 reserved Ground (0V) Trig1I Trigger Input #1 Trig2I Trigger Input #2 Trig3I Trigger Input #3...
Page 246: Monitordiode
4.12.7 Monitordiode The monitordiode is placed in the excitation ray path of the LSM 510 behind the beam splitter combining the visible and the uv ray path and in front of the main beam splitter. Therefore it allows for checking the laser input in terms of power and noise.
Page 247 OPERATION LSM 510 Annex Change to the "Scan Control" window and press <Cont.>; the system will scan with the diode as a channel. Choose the right amplification of the signal obtained by using the special neutral density filters in front of the diode or / and by using the setting of the Amplifier Gain and Offset value. ("Scan Control"...
Page 248 OPERATION Annex LSM 510 Examples of application: Checking the laser power This function is not automized so far. To qualitatively measure the laser power the diode can be used in such a way, that the graylevel obtained in the Line Scan mode at a certain setting of the whole system is stored as a text overlay together with the image (manually done by user).
Page 249 OPERATION LSM 510 Annex To use the monitordiode for ratio application the following steps have to be done: Activate the ratio channel R1 or R2 in <Ratio Settings> of the menu <Configuration Control> in addition to the monitor diode channel (ChM-1) and one PMT channel.
Page 250 OPERATION Annex LSM 510 The following image is an example of the reduction of correlated noise. The low frequency noise has been generated artificially. Fig. 4-179 The image in the upper left corner shows the PMT image plus noise, the image beneath this (upper right corner) shows the signal of the diode expanded to 512x512 pixels (noise without object information).
Page 251 OPERATION LSM 510 Annex The single steps to find the right setting of all the parameters to be set are listed in the following: Activate the <Range Indicator> Adjustment of Amplifier Offset: the Offset of the PMT channel and diode channel have to fit to each other to guarantee the best noise reduction.
Page 252 OPERATION Annex LSM 510 Amplifier Gain The diode signal is set to the right range (graylevel between 50 and 200 - 8bit image / 750 and 3500 - 12 bit image) with the help of gray filters and amplifier gain. The use of a lower filter density should be priorized against the use of a high gain value.
Page 253 OPERATION LSM 510 Annex Gain and offset in Ratio channel If the setting of the PMT channel is finished, the range of the ratio channel is adjusted by the parameters in the corresponding formalism. There are three types of formulas offered, when the button <R1-1> is pushed.
Page 254 OPERATION Annex LSM 510 Default settings are 150 and 3000 respectively. With the help of the range indicator the default value is changed until there is no pixel overflow anymore (‘red pixels’) Any new value can be set by hand typing and pressing the <ENTER> key while the scan is running.
Page 255 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Contents CHAPTER 5 3D FOR LSM 510 CONTENTS Page 3D FOR LSM 510 .......................5-3 Overview and Explanations..................5-3 5.1.1 The Image Sequence ....................5-3 5.1.2 The Image Properties....................5-4 5.1.3 Memory Usage......................5-4 User Interface ......................5-5 5.2.1...
Page 256 INTRODUCTION TO LASER SCANNING MICROSCOPY Contents LSM 510 B 40-051 e 07/98...
Page 257: For Lsm 510
The Image Sequence (0, 0, 0) Voxel The "3D for LSM" handles image sequences generated by the Zeiss LSM software. This can be Single slice with three-dimensional image data or a time sequence single channel of two-dimensional images (slices). Each slice (as well as the sequence) can consist of up to eight channels.
Page 258: The Image Properties
INTRODUCTION TO LASER SCANNING MICROSCOPY Overview and Explanations LSM 510 Image sequences can consist of several channels. Most functions and the Display window are providing buttons to select all or a subset of channels stored in the selected image sequence. The Output image sequence will only get those channels which are selected on the input side.
Page 259: User Interface
INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 User Interface User Interface 5.2.1 Introduction This section describes the following main components of the system: Main window Main window with the Menu, the Tool bar and Gallery. All general system functions are located here.
Page 260: Display Window
INTRODUCTION TO LASER SCANNING MICROSCOPY User Interface LSM 510 Display window This window is used to display image sequences. Display window Fig. 5-3 Display window Dialog boxes All dialog boxes provide three buttons. Pressing the OK button executes the function with the defined parameters and closes the dialog window. Selecting the Cancel button does not execute the function, restores the parameters, and closes the dialog window.
Page 261 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 User Interface 5.2.2 Main Window The Main window includes: the Menu the Tool bar and the Gallery File Menu Open Image Opens a file selector dialog to load an image sequence. Save Image As Opens a file selector to save an image or image sequence.
Page 262 INTRODUCTION TO LASER SCANNING MICROSCOPY User Interface LSM 510 Edit Menu Copy Copies the contents of the Display window to the clipboard. Edit Channels Allows to add or to remove channels to a single or multichannel image. Delete All Images Deletes all images and image sequences from the memory.
Page 263 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 User Interface View Menu Set Channel Colour The colour and the weight of the single channels can be defined. Properties The properties of the image (e.g. scaling, use laser etc.) are displayed. Render Calculates 3D reconstructions of an image sequence (Surface, Alpha).
Page 264 INTRODUCTION TO LASER SCANNING MICROSCOPY User Interface LSM 510 Gallery The Gallery is used as an overview of the images available in memory and their contents. It is located just below the Tool bar. Each small image represents a sequence. The middle slice of each image sequence is shown.
Page 265 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 User Interface To start the automatic animation of an image sequence start the Player tool by clicking on the button . The colour selection for the channels can be activated by clicking on the button .
Page 266 INTRODUCTION TO LASER SCANNING MICROSCOPY User Interface LSM 510 Control Element of the Player The three arrow shaped controls on the scale show the start slice and the currently displayed sequential image. The values (positions) can be changed using the mouse. Press and hold the left mouse button and move the pointer to the desired position.
Page 267 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 User Interface Set Channel Colour This function sets the colour and weight for the channels. Fig. 5-5 Each image sequence can get its own colour definitions. All functions will inherit the colour definition from the Input sequence to the Output sequence.
Page 268: Functions
5.3.1 Functions in the File Menu Open Image This function reads a Zeiss LSM 510 (*.lsm), Zeiss LSM TIFF (*000.tif) or Carl Zeiss Vision (*0.img) image sequence from a disk or network drive. Fig. 5-6 The individual files of a Zeiss TIFF image sequence are read and saved as an image sequence in image memory.
Page 269 LSM 510 Functions Carl Zeiss Vision image sequences must have a number digit at the end of the base filename. They are used to indicate the different channels in a multichannel sequence. The numbering starts with zero (0). If a sequence is saved in the Carl Zeiss Vision format the numbers are generated automatically. To load such an image sequence "KE Images (*0.img)"...
Page 270 The directories of the current drive are listed in the Directories list box. Use the Drives list box to choose a different drive. Use the list box Files of Type to select the image format. Currently the LSM 510 image format (*.lsm) and the Carl Zeiss Vision file format "KE Images (*0.img)" is supported.
Page 271 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions The content of the Gallery is shown in the Input section. The selection of the sequence to save is done by highlighting one of the provided names or by drag and drop from the Gallery.
Page 272 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Print This function prints the current Display window contents. The standard Windows print dialog is opened. Before the execution of this function any image or image sequence can be selected to be displayed.
Page 273: Functions In The Edit Menu
INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions 5.3.2 Functions in the Edit Menu Copy This function copies the current Display window contents to the clipboard. No dialog is shown. Before the execution of this function any image or image sequence can be selected to be displayed.
Page 274 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Edit Channels This function allows to add or to remove channels to a single or multichannel image. On the Add Channel tab sheet the channels of (different) Input sequences can be defined to add (combine) channels to an Output sequence.
Page 275 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions On the Delete Channel tab sheet channels of the Input 1 image sequence can be selected to delete channels. Fig. 5-10 This operation might save time and memory for further processing if not all channels are needed.
Page 276: Functions In The Process Menu
INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 5.3.3 Functions in the Process Menu Arithmetics - Add This function adds two image sequences. Fig. 5-11 The Add tab sheet of the Arithmetics dialog window must be selected. If one or both input sequences are multichannel sequence, any number or combination can be selected.
Page 277 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions This function adds the two image sequences Input 1 and Input 2 voxel by voxel and generates the image sequence Output. Note that a resulting grey value may be greater than 255 (4095). The...
Page 278 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Arithmetics - Subtract This function subtracts two image sequences. Fig. 5-12 The Subtract tab sheet of the Arithmetics dialog window must be selected. If one or both input sequences are multichannel sequence, any number or combination can be selected.
Page 279 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions 1 - Wrap No normalization - the grey values are displayed modulo 256 (4096). If the result is less than 0, the value 256 (4096) is added to it. 2 - Clip Negative values are set to 0.
Page 280 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Contrast - Interactive This function allows interactive changes of the contrast of an image sequence. Fig. 5-13 The Interactive tab sheet of the Contrast dialog window must be selected. 5-26 B 40-051 e 07/98...
Page 281 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions A grey value range of the Input image sequence is scaled to another range in the Output image sequence. Both ranges can be edited interactively. This function is used to achieve a better view of an image sequence, or to scale a range of grey values to single value for a special coding in an image sequence.
Page 282 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Entering a new value in the appropriate text boxes, clicking on the buttons or using the arrow keys from the keyboard. To alter the values within the histogram move the mouse pointer over one of the three coloured lines until the shape changes.
Page 283 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Contrast - Automatic This function scales the grey values of an image sequence to the maximum possible range. Fig. 5-14 The Automatic tab sheet of the Contrast dialog window must be selected.
Page 284 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Output defines the name of the result sequence. It will get only those channels which are chosen by the Input parameter. The buttons labeled with 8 and 12 define the grey value (intensity) resolution in bit.
Page 285 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Contrast – Linearize This function scales a range of grey values of an image sequence to equal area fractions in the histogram. Fig. 5-15 The Linearize tab sheet of the Contrast dialog window must be selected.
Page 286 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 The Output histogram shows the resulting histogram. The horizontal axis represents the grey values from 0 to 255. The vertical axis represents the pixel count. The vertical scale of the histogram is set using the scroll bar.
Page 287 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Smooth (Gauss) This function performs a Gauss filter. Fig. 5-16 The noise in the image sequence is reduced, the edge shape is nearly unchanged, local maxima are leveled, the dynamic range is reduced.
Page 288 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Morphology The following four functions perform basic operations of mathematical morphology on image sequences. Fig. 5-17 As generalization of the morphology of two-dimensional images to three dimensions the structural elements are small volumina.
Page 289 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions The input image sequence is analyzed voxel by voxel with a selected shape (Shape). The voxel to be analyzed is always the central voxel of the shape. The shape type determines which neighboring voxels are used to compute the resulting voxel.
Page 290 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Sequential image: Volume view: Cube cross shape: created through application of "cube" and "cross" one after the other. For regions (voxels) that are at the edge of the image sequence, it assumed for erosion that there are white voxels with a grey value of 255 (4095) outside the edge.
Page 291 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions The result of erosion and dilation is called opening. On the one hand, this maintains to some extent the original size of the regions while not losing the smoothing effect of erosion on the image. This name stands for the operation of reducing convex bulges in the contour of the region.
Page 292 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Morphology - Erode This function erodes structures in an image sequence. Fig. 5-20 In the Morphology dialog window, the tab sheet Erode must be selected. Erosion makes bright regions smaller on a dark background. It also results in separation of thin connections between regions.
Page 293 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Parameters: Input Input image sequence Output Resulting image sequence Shape Shape used 1 - cross 2 - cube 3 - cube cross Count Number of recursive operations Grey Morphology 0 - Distinguish between 0 and non 0 only...
Page 294 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 The following shapes (numbered 1 to 3 from left to right) are available: If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result Output will be a binary sequence.
Page 295 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Morphology - Open This function carries out an opening. Fig. 5-22 In the Morphology dialog window, the tab sheet Open must be selected. This function carries out an erosion followed by a dilation. For the most part, the opening maintains the original size of the regions.
Page 296 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Parameters: Input Input image sequence Output Resulting image sequence Shape Shape used 1 - cross 2 - cube 3 - cube cross Count Number of recursive operations Grey Morphology 0 - Distinguish between 0 and non 0 only...
Page 297 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Morphology - Close This function carries out a closing. Fig. 5-23 In the Morphology dialog window, the tab sheet Close must be selected. This function carries out a dilation followed by an erosion. For the most part, the closing maintains the original size of the regions.
Page 298 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Parameters: Input Input image sequence Output Resulting image sequence Shape Shape used 1 - cross 2 - cube 3 - cube cross Count Number of recursive operations Grey Morphology 0 - Distinguish between 0 and non 0 only...
Page 299 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Segment - Interactive This function carries out a grey value segmentation by means of thresholding. Fig. 5-24 The Interactive tab sheet of Segment dialog window must be selected. Segmentation is especially used to generate binary regions. These are required for the measurement.
Page 300 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 The Green and Blue/Red option buttons of the parameter Colour determine whether the voxels within (Green) or outside (Blue/Red) of the grey value interval [L, H] are displayed with the corresponding colour.
Page 301 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Segment - Automatic The function carries out an automatic grey value segmentation by means of thresholding. Fig. 5-25 The Automatic tab sheet of the Segment dialog window must be selected. Segmentation is especially used to generate binary regions.
Page 302 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 If Blue/Red is selected, the voxels with grey values within the interval Low, High remain unchanged. Voxels with grey values less than Low are highlighted in blue; those with grey values higher than High are highlighted in red.
Page 303 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Boolean - And This function carries out a bit-by-bit And calculation for the image sequences Input 1 and Input 2. Fig. 5-26 The And tab sheet of the Boolean dialog window must be selected.
Page 304 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Boolean - Or This function carries out a bit-by-bit Or calculation for the images Input 1 and Input 2. Fig. 5-27 The Or tab sheet of the Boolean dialog window must be selected.
Page 305 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Boolean - Xor This function carries out a bit-by-bit Xor calculation for the images Input 1 and Input 2. Fig. 5-28 The Xor option button of the Function option group in the Boolean dialog window must be selected.
Page 306 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Boolean - Not This function carries out a bit-by-bit negation of an image. Fig. 5-29 The Not tab sheet of the Boolean dialog window must be selected. If Input is a multichannel sequence any number or combination can be selected.
Page 307 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Boolean - Mask This function masks a grey value image sequence. Fig. 5-30 The Mask tab sheet of the Boolean dialog window must be selected. This function modifies the Output image sequence depending on the mask image sequence used.
Page 308 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Scrap This function deletes or selects objects in a specified size range. Fig. 5-31 The operation deletes or selects objects on the basis of their total volume in voxels. Objects with a volume within the range MinVolume to MaxVolume are effected.
Page 309 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Fill Holes This function fills holes in all objects. Fig. 5-32 All holes in objects are filled by this operation. Holes are structures, which have a grey value of 0 and are surrounded completely by voxels with a grey value not equal to 0.
Page 310: Functions In The View Menu
INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 5.3.4 Functions in the View Menu Render - Surface This function displays an image sequence according to the gradient shading method. Fig. 5-33 The Surface tab sheet of the Render dialog window must be selected.
Page 311 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Method The Input sequence defines the data to be reconstructed. If it is a multichannel sequence one or all channels can be selected for the reconstruction. Output sets the name of the result image (sequence). If the sequence exists it is overwritten. Pressing the button New will generate a new name (number).
Page 312 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Use the parameter Reflection to control the ratio of diffuse and reflective brightness components, i.e., the overall basic brightness compared with the highlights. When the value of Reflection is low, the highlights predominate; when the values are high, the region appears to be uniformly illuminated and the highlights are not so pronounced.
Page 313 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Render - Surface: Method Description This method displays the surface of structures in the Input sequence shaded as if a light illuminated it. The position of the light is behind the view point with parallel rays in the direction of the sequence.
Page 314 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Render - Alpha This function displays an image sequence according to the alpha rendering method. Fig. 5-34 The Alpha tab sheet of the Render dialog window must be selected. One or more reconstructions of the input image sequence are computed according to the alpha rendering method.
Page 315 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Method The Input sequence defines the data to be reconstructed. If it is a multichannel sequence one or all channels can be selected for the reconstruction. Output sets the name of the result image (sequence). If the sequence exists it is overwritten. Pressing the button New will generate a new name (number).
Page 316 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Application 1. This method can be applied, if the structures in the Input sequence are unsharp so that objects are poorly defined by their grey value. 2. In this case, the Opacity Table is defined as a ramp. Low grey values have weight 0 to suppress the background voxels.
Page 317 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Render - Alpha: Method Description Each Output pixel is a weighted sum of the Input voxels along a ray in view direction through the Input sequence. Each Input voxel has an opacity value, dependent only on its grey value. The opacity values are defined by the parameters Threshold, Ramp, and Max.
Page 318: Functions In The Measurement Menu
INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 5.3.5 Functions in the Measurement Menu Measurement Concept Measurement is based on regions (objects) in three-dimensional space. Segmenting an image sequence generates these. The image segmentation process produces a mask image that defines the region.
Page 319 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Measurement Process The measurement process consists of three steps: region definition, checking of the validity of the regions, and feature calculation. Region definition: - Automatically from the mask image Region validation check depends on:...
Page 320 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Automatic Object Measurement – Object Features A measurement feature describes a region characterized by a number (e.g. volume, area or a densitometrical statistic). The features can be selected on the Object Features and Volume Features tab sheets.
Page 321 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions The dialog shows two lists. One shows the Available Features as groups (on the left). The other one shows the Selected Features. Double-clicking on items of the left list will add the Selected Features to the right list.
Page 322 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Object Features (geometric) If Object Features are selected, one set of measurement data is calculated for each object. Group Name Name Description Volume Volume Volume of the object. Volume Filled VolumeF Volume of the filled object.
Page 323 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Object Features (densitometric) Group Name Name Description Mean Densitometric MeanD Densitometric mean value of an object. Standard Deviation StdD Standard deviation of the densitometric values of an object. Densitometric Minimum MinD Minimum grey value of an object.
Page 324 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Automatic Object Measurement - Volume Features A measurement feature describes a region characterized by a number (e.g. volume, area, or a densitometrical statistic). The features can be selected on the Object Features and Volume Features tab sheets.
Page 325 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Parameters: Available Features List of available object features Selected Features List of selected object features Select All Select all available object features for measurement Remove All Remove all object features from the selected features list...
Page 326 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 Volume Features (geometric) The volume-related measurement generates one measured value per image sequence. The following table contains the predefined volume characteristics. Group Name Name Description Count VolCount Number of regions measured. Volume VolVolume Total volume of all regions.
Page 327 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Automatic Object Measurement - Condition The measurement conditions are used to limit the objects to be evaluated (e.g. only objects with defined minimum value). All objects are tested against the defined conditions. If the conditions are fulfilled the feature values are written to the data table.
Page 328 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 The list on the very left at the dialog shows all the measurement Features. The second list provides the comparison Operators and the next Numbers to define a value. This gives the possibility to compose an expression to test a feature value against a constant value.
Page 329 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 Functions Automatic Object Measurement - General This function carries out an automatic measurement and labeling. Measured Object Features Measured Volume Features Fig. 5-40 The regions must be defined by an image sequence Mask Image (the objects must be separated from one another by black voxels with the grey value 0).
Page 330 INTRODUCTION TO LASER SCANNING MICROSCOPY Functions LSM 510 A single object of interest can be visualized. Clicking on a specific row in the data grid chooses the object. By selecting a row in the data grid a new image is created with the object of interest visualized.

Zeiss LSM 510 Operating Manual

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