Page 1 March 2006...
Page 2 LSM 510 DuoScan Carl Zeiss LSM 510 META DuoScan 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 META Carl Zeiss How to make best use of the LSM 510 and LSM 510 META operating instructions: This operating manual also includes the instructions for the LSM 510 META DuoScan and the LSM 510 DuoScan systems.
Page 4 Here you will find an introduction to Laser Scanning Microscopy, with an explanation of the principles of confocal imaging. The section also outlines the ways to present LSM image series in three dimensions, and introduces you to the performance features of your LSM 510 or LSM 510 META.
Page 5: Table Of Contents
LSM 510 NOTES ON DEVICE SAFETY LSM 510 META General Carl Zeiss CHAPTER 1 NOTES ON DEVICE SAFETY CONTENTS Page NOTES ON DEVICE SAFETY .....................1-2 General..........................1-2 Warning and Information Labels ..................1-3 Regulations ........................1-11 Notes on Setting up the Microscope System ..............1-12 Power Requirements ......................1-14...
Page 6: Notes On Device Safety
3B and 4. If equipped with a Ti:Sa Laser (see list in section 1.6), the LSM 510 and LSM 510 META are devices that belong to laser hazard class 4. WHO recommendations concerning health and industrial protection when handling laser devices must be observed.
Page 7: Warning And Information Labels
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. You will receive a free replacement.
Page 8 Carl Zeiss Warning and Information Labels LSM 510 META DuoScan T IV J E C F L E Fig. 1-1 Warning and information labels on the Axiovert 200 M microscope with the LSM 510 META scanning module B 45-0021 e 03/06...
Page 9 LSM 510 NOTES ON DEVICE SAFETY LSM 510 META Warning and Information Labels Carl Zeiss Fig. 1-2 Warning and information labels on the Axio Imager.Z1 microscope with LSM 510 META scanning module 03/06 B 45-0021 e...
Page 10 NOTES ON DEVICE SAFETY LSM 510 DuoScan Carl Zeiss Warning and Information Labels LSM 510 META DuoScan Fig. 1-3 Warning and information labels on the Axioskop 2 FS microscope with the LSM 510 META scanning module B 45-0021 e 03/06...
Page 11 LSM 510 META Warning and Information Labels Carl Zeiss Fig. 1-4 Warning and information labels on LSM DuoScan (systems LSM 510 DuoScan or LSM 510 META DuoScan only) Fig. 1-5 Warning and information labels on the system electronic rack 03/06...
Page 12 NOTES ON DEVICE SAFETY LSM 510 DuoScan Carl Zeiss Warning and Information Labels LSM 510 META DuoScan Fig. 1-6 Warning and information labels on laser components (page 1) B 45-0021 e 03/06...
Page 13 LSM 510 NOTES ON DEVICE SAFETY LSM 510 META Warning and Information Labels Carl Zeiss Fig. 1-7 Warning and information labels on laser module LSM 5 LIVE 03/06 B 45-0021 e...
Page 14 NOTES ON DEVICE SAFETY LSM 510 DuoScan Carl Zeiss Warning and Information Labels LSM 510 META DuoScan R I S xi m te r ss u p re p. s. te r R I S Fig. 1-8 Warning and information labels on laser components (page 2)
Page 15: Regulations
⇒ The LSM 510 and LSM 510 META have been designed, built and tested in conformity with the following regulations and guidelines: DIN EN 61010-1 (IEC 601010-1) "Safety requirements for electrical equipment for measurement,...
Page 16: Notes On Setting Up The Microscope System
The LSM 510 or LSM 510 META laser scanning microscope is delivered in several crates. The LSM 510 and LSM 510 META 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 17 By establishing a corresponding workplace environment, please ensure that the formation of electrostatic charges of electronic components is avoided. To avoid vibrations during operation, the LSM 510 and LSM 510 META should only be operated in conjunction with the system table (vibration damping).
Page 18: Power Requirements
LSM 510 META DuoScan Power Requirements The LSM 510 and LSM 510 META comes with a mains power supply cord and plug, either CEE red (3/N/PE 400/230V/16A), or NEMA L 14-30P (2/N/Ground 120/240V/30A), and with the matching mains socket outlet.
Page 19: Notes On Handling The Laser Components
Only personnel which has been instructed on laser safety is allowed to operate the system. The following laser types are currently intended for use in the LSM 510 and LSM 510 META. The use of any other lasers as the ones listed below is not authorized.
Page 20 LSM 510 DuoScan Carl Zeiss Notes on Handling the Laser Components LSM 510 META DuoScan • If necessary - insofar as specified by law - inform the laser protection officer before commissioning the laser. • The laser modules are equipped with a key-interlock.
Page 21: Physical Dimensions
Active anti-vibration table (NLO) for Mai Tai Laser or Chameleon Active anti-vibration table (NLO) for Verdi Mira or Millenia Tsunami Laser Scanning Module LSM 510 Scanning Module LSM 510 META 30.5 Scanning Module LSM DuoScan Microscope Laser Module RGB Laser Module, UV...
Page 22: Notes On Handling The Computer And Data Media
Do not place the disc in any place where it is exposed to direct sunlight or high temperatures. Backup your data on a regular basis. Do not install any other software without talking to your Carl Zeiss representative. Never turn your computer off before you have terminated the LSM program and run down the WINDOWS XP operating system.
Page 23: Notes On Care, Maintenance And Service
Any such action will render all warranty claims null and void and also laser safety is no longer warranted. You are well advised to arrange a service agreement with your nearest Carl Zeiss representative to guarantee perfect functioning of the microscope system in the long term.
Page 24: User Interface
These interlock devices must not be manipulated. Other interfaces which are not described here are service interfaces and are only to be operated by authorized Carl Zeiss service personnel. The following devices can be mounted and dismounted by the user: −...
Page 25: Mounting And Dismounting The Scan Head
LSM 510 NOTES ON DEVICE SAFETY LSM 510 META User Interface Carl Zeiss In case the system is not operating following the removal or attachment of any device on a port with safety interlock check again the connection of the Contact ring to the Sensor ring.
Page 26 LSM 510 META DuoScan Moving the scan heads between Axiovert 200 M, Axioskop 2FS and Axio Imager.Z1: • LSM 510 META scan head: Remove the blue cover from the back of the scan head (Fig. 1-13 and Fig. 1-14). ⇒...
Page 27 LSM 510 NOTES ON DEVICE SAFETY LSM 510 META User Interface Carl Zeiss • Loosen the screws on the port to the microscope for Axiovert 200 M (Fig. 1-17/1) or to the tube on Axio Imager.Z1 and Axioskop 2 FS (Fig.
Page 28 For NLO systems being coupled to a Chameleon or Verdi Mira Laser from Coherent using the beam housing kit designed for LSM 510, the beam tube connecting the periscope and the scan head has to be removed before the scan head can be dismounted. Perform the following steps for the removal of the tube: •...
Page 29 LSM 510 NOTES ON DEVICE SAFETY LSM 510 META User Interface Carl Zeiss To ensure functioning of the system and laser safety the following connections have to be changed: The connection of the microscope to the safety interface of the system is located either on the additional Safety-Box (Axioskop 2 FS , Fig.
Page 30 NOTES ON DEVICE SAFETY LSM 510 DuoScan Carl Zeiss User Interface LSM 510 META DuoScan The plug for the main connection of the microscope to the electronics is situated on the rear side of the electronics rack. It is either of the four CAN connections shown in Fig.
Page 31 LSM on passive anti vibration table (one microscope, upright or inverted): 2,2 m x 4,2 m ..2-4 2.1.4 LSM with Ar UV Laser ......................2-5 2.1.5 LSM 510 or LSM 510 META equipped with LSM DuoScan UV ..........2-7 2.1.6 LSM prepared for Two Photon Laser (NLO) .................2-8 Power Requirements ......................2-10 Physical Dimensions ......................2-14...
Page 32: Lsm 510 And Lsm 510 Meta - Setup Requirements
LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss Space Requirements LSM 510 META DuoScan LSM 510 AND LSM 510 META - SETUP REQUIREMENTS Space Requirements LSM on large system table (one microscope, upright or inverted): 2 m × 3,50 m 2.1.1...
Page 33: Lsm On Small System Table (One Microscope, Upright Or Inverted): 2 M X 3,5 M
LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META Space Requirements Carl Zeiss 2.1.2 LSM on small system table (one microscope, upright or inverted): 2 m x 3,5 m Fig. 2-2 Space requirements for LSM on small system table...
Page 34: Lsm On Passive Anti Vibration Table (One Microscope, Upright Or Inverted): 2,2 M X 4,2 M
LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss Space Requirements LSM 510 META DuoScan 2.1.3 LSM on passive anti vibration table (one microscope, upright or inverted): 2,2 m x 4,2 m Fig. 2-3 Space requirements for LSM on Passively Damped Anti Vibration Table (system table with breadboard).
Page 35: Lsm With Ar Uv Laser
LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META Space Requirements Carl Zeiss 2.1.4 LSM with Ar UV Laser We recommend placing the cooling unit of the Ar laser (UV) in a separate room to prevent heat accumulation and vibration.
Page 36 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss Space Requirements LSM 510 META DuoScan Two microscopes: 3 m x 3 m Fig. 2-5 Space requirements for LSM with two microscopes and AR UV Laser...
Page 37: Lsm 510 Or Lsm 510 Meta Equipped With Lsm Duoscan Uv
LSM 510 META Space Requirements Carl Zeiss 2.1.5 LSM 510 or LSM 510 META equipped with LSM DuoScan UV Fig. 2-6 Space requirements for LSM equipped with LSM DuoScan and AR UV Laser (measurements in mm) The System Electronic Rack contains all electronics for control of the hardware components of the LSM system, the power supply for the microscope, the scanning unit and the Laser Module RGB equipped with the choice of lasers.
Page 38: Lsm Prepared For Two Photon Laser (Nlo)
LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss Space Requirements LSM 510 META DuoScan 2.1.6 LSM prepared for Two Photon Laser (NLO) 2.1.6.1 Coherent "Chameleon" or Spectra Physics “Mai Tai”. Directly-coupled to upright or inverted Microscope: 2,5 m x 4 m Fig.
Page 39 LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META Space Requirements Carl Zeiss 2.1.6.2 Coherent "Verdi Mira" or Spectra Physics “Millenia Tsunami”. Directly-coupled with upright or inverted Microscope: 3,5 m x 3,5 m Fig. 2-8 Space requirements for Two Photon Laser and Large NLO System Table.
Page 40: Power Requirements
LSM 510 META DuoScan Power Requirements The LSM 510 and LSM 510 META comes with a mains power supply cord and plug, either CEE red (3/N/PE 400/230V/16A), or NEMA L 14-30P (2/N/Ground 120/240V/30A), and with the matching mains socket outlet.
Page 41 Carl Zeiss Fig. 2-9 Power connector for LSM 510 / LSM 510 META and Laser Module V. Free/reserve outlets may be used to supply power to additional equipment. No more than 1 A can be provided by each outlet. (Scheme is turned 90° to real system.)
Page 42 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss Power Requirements LSM 510 META DuoScan Fig. 2-10 Power connector for Laser Module RGB (Scheme is turned 90° to real system.) 2-12 B 45-0021 e 03/06...
Page 43 LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META Power Requirements Carl Zeiss Key-interlock Laser ON/OFF Door interlock interface Fig. 2-11 Key-interlock Laser ON/OFF and interface for connection of door interlock The door interlock interface is covered with a green plug to bypass a door interlock.
Page 44: Physical Dimensions
LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss Physical Dimensions LSM 510 META DuoScan Physical Dimensions Length (cm) Width (cm) Height (cm) Weight (kg) Large system table Small system table Passively damped anti-vibration table...
Page 45: Dimension Of Shipment Crates
LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META Dimensions ... / Environmental Requirements Carl Zeiss Dimension of Shipment Crates Crate containing Length (cm) Width (cm) Height (cm) Weight (kg) Large system table Small system table...
Page 46: Vibrations
Microscopes Inverted Axiovert 200 M BP or SP Upright Axioskop 2 FS Upright Axio Imager.Z1 Upright Axio Imager.M1 All ICS objectives from Carl Zeiss and their accessories can be accommodated. Z motor DC servomotor, opto-electronically coded Least Z interval: 50 nm (Axiovert 200 M BP or SP) 100 nm (Axioskop 2 FS 10 nm (Axio Imager.Z1)
Page 47: Scanning Module Lsm 510 And Lsm 510 Meta
LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META Scanning Module Carl Zeiss Scanning Module LSM 510 and LSM 510 META Scanners 2 individually driven galvanometric scanners Up to ~5 frames/sec (512 × 512 pixels) Scanning speed Max.
Page 48: Laser Module Rgb (458, 477, 488, 514, 543 Or 561,594, 633 Nm)
LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss Laser Modules LSM 510 META DuoScan 2.10 Laser Module RGB (458, 477, 488, 514, 543 or 561,594, 633 nm) Single-mode polarization preserving fiber Laser beam attenuation for all lasers by VIS-AOTF...
Page 49: System Overview Lsm 510 Meta
LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META System Overview LSM 510 META Carl Zeiss 2.14 System Overview LSM 510 META 03/06 B 45-0021 e 2-19...
Page 50 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss System Overview LSM 510 META LSM 510 META DuoScan 2-20 B 45-0021 e 03/06...
Page 51: System Overview Lsm 510 Meta Duoscan
LSM 510 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 META System Overview LSM 510 META DuoScan Carl Zeiss 2.15 System Overview LSM 510 META DuoScan 03/06 B 45-0021 e 2-21...
Page 52 LSM 510 AND LSM 510 META - SETUP REQUIREMENTS LSM 510 DuoScan Carl Zeiss System Overview LSM 510 META DuoScan LSM 510 META DuoScan 2-22 B 45-0021 e 03/06...
Page 53 INTRODUCTION TO LASER SCANNING MICROSCOPY ...........3-2 Principle of Laser Scanning Microscopy................3-2 Three-Dimensional Presentations of LSM Image Stacks ............3-3 Optical Diagram of the LSM 510 and LSM 510 META (Schematic) ........3-4 Performance Features of the LSM 510 and LSM 510 META ..........3-5 3.4.1 Optical and Mechanical Aspects ..................3-5...
Page 54: Introduction To Laser Scanning Microscopy
INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 DuoScan Carl Zeiss Principle of Laser Scanning Microscopy LSM 510 META DuoScan 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;...
Page 55: Three-Dimensional Presentations Of Lsm Image Stacks
LSM 510 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 510 META Three-Dimensional Presentation of LSM Image Stacks Carl Zeiss Three-Dimensional Presentations of LSM Image Stacks One of the advantages of the LSM technique is that it can present structures in three dimensions. This opens up many ways to process images.
Page 56: Optical Diagram Of The Lsm 510 And Lsm 510 Meta (Schematic)
LSM 510 DuoScan Carl Zeiss Optical Diagram of the LSM 510 LSM 510 META DuoScan Optical Diagram of the LSM 510 and LSM 510 META (Schematic) Fig. 3-2 Optical path, schematic (4-channel configuration) AOTF Acousto Optical Tunable Filter Dichroic Beam Combiner...
Page 57: Performance Features Of The Lsm 510 And Lsm 510 Meta
LSM 510 META Performance Features of the LSM 510 … Carl Zeiss Performance Features of the LSM 510 and LSM 510 META 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.
Page 58: Microscope Equipment Of The Lsm 510 And Lsm 510 Meta System
This will prevent cross talk between detection channels. The LSM 510 and LSM 510 META can be combined with a second scanner, the LSM DuoScan. Any of the above mentioned microscopes can be used. The LSM DuoScan can be equipped either with a UV Laser or a 405 nm laser both also together with a 488 and/or 532 nm laser.
Page 59 (6) Photo equipment The stand does not feature an integrated SLR-port, but microscope cameras as described in the Axiovert 200 M and LSM 510 / LSM 510 META operation manual can be used. (7) TV adaptation The TV port at the side and the tubes can be used as described in the Axiovert 200 M operation manual.
Page 60: Computer Hardware And Software
The software of the LSM 510 and LSM 510 META have 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 61 IMPORTANT NOTES FOR CHAPTER 4 Bleach series When doing bleach series on LSM 510 DuoScan systems, the time stamps and bleach markers indicated in the images will depend on the start position of the scanners in the image frame. Since these are not identical for the imaging and the photomanipulation scanners, sometimes imaging seems to start too early after a bleach event.
Page 62 IMPORTANT NOTES FOR CHAPTER 4 LSM 510 DuoScan Carl Zeiss LSM 510 META Duo Scan 4-II B 45-0021 e 03/06...
Page 63 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Purpose Carl Zeiss CHAPTER 4 OPERATION IN EXPERT MODE CONTENTS Page OPERATION IN EXPERT MODE ................4-9 Purpose ........................... 4-9 4.1.1 Software ........................... 4-9 4.1.2 Windows and Window Elements ..................4-10 4.1.3...
Page 64 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Purpose LSM 510 META DuoScan Acquire Menu ........................4-38 4.5.1 Laser Control ........................4-39 4.5.1.1 Opening / Closing the Laser Control Window..............4-39 4.5.1.2 Function Description ......................4-39 4.5.1.3 Settings..........................4-40 4.5.2 Microscope Control......................4-41 4.5.2.1 Open the Microscope Control Window ................4-41 4.5.2.2...
Page 65 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Purpose Carl Zeiss 4.5.8 Stage ..........................4-122 4.5.8.1 Open / Close the Stage and Focus Control Window ............4-123 4.5.8.2 Focus Position Panel ...................... 4-123 4.5.8.3 Stage Position Panel ...................... 4-124 4.5.8.4...
Page 66 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Purpose LSM 510 META DuoScan 4.6.12 Ion Concentration ......................4-149 4.6.12.1 Open / Close the Ion Concentration Window ..............4-149 4.6.12.2 Function Description ......................4-149 4.6.12.3 Single Wavelength Dyes – Offline Calibration ..............4-150 4.6.12.4 Ratiometric Dyes ......................4-151 4.6.12.5 Ratiometric Dyes - Online Ratio ..................4-151...
Page 67 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Purpose Carl Zeiss 4.8.4.2 Distance Macro - Distance.lvb ..................4-184 4.8.4.3 Profile Macro – Profile.lvb....................4-185 4.8.4.4 Bleach Macro – Bleach.lvb..................... 4-185 4.8.4.5 Excitation Fingerprinting....................4-186 4.8.4.6 Concatenate Images ..................... 4-190 4.8.4.7...
Page 68 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Purpose LSM 510 META DuoScan 4.10.3.3 Collimator Panel......................4-239 4.10.3.4 Pinhole and Collimator Adjustment ................4-240 4.10.4 Set Find..........................4-242 4.10.5 Spline..........................4-242 4.10.6 Camera..........................4-243 4.10.7 HWAdmin........................4-243 4.10.8 Test Grid ........................4-243 4.11 Window Menu......................4-244 4.11.1 Full Screen ........................4-244...
Page 69 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Purpose Carl Zeiss 4.13.16 Display - Split xy ......................4-268 4.13.17 Display - Ortho......................4-269 4.13.17.1 Ortho - Select Function ....................4-270 4.13.17.2 Ortho - Distance Function ..................... 4-271 4.13.17.3 Ortho - 2D DeConVolution Function ................4-272 4.13.18...
Page 70 4.14.1.3 Scan Speed, Scan Average and Pixel Depth ..............4-352 4.14.2 Multiple-channel ......................4-353 4.14.2.1 Requirements.........................4-353 4.14.2.2 Image Optimization......................4-354 4.15 Extension of the LSM 510 or LSM 510 META with LSM DuoScan ......4-355 4.16 Shut-Down Procedure....................4-356 4.16.1 Exiting the LSM Program ....................4-356 4.16.2 Shut Down the WINDOWS Operating System..............4-357...
Page 71: Operation In Expert Mode
OPERATION IN EXPERT MODE Purpose This chapter describes the operation of the LSM 510 and LSM 510 META Laser Scanning Microscopes, also when equipped with LSM DuoScan, exemplified by typical applications in conjunction with the LSM 5 software and its graphic user environment.
Page 72: Windows And Window Elements
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Purpose LSM 510 META DuoScan 4.1.2 Windows and Window Elements Window element Description / Explanation Window (e.g.: Laser Control window) − Window displayed after activation of a function button (e.g.: Laser button in the toolbar of the Expert Mode).
Page 73: Convention For The Text In This Manual
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Purpose Carl Zeiss 4.1.3 Convention for the Text in this Manual All the originally used terms of the software interface, e.g. − names of windows, − panels, − input boxes, − list / selection boxes, −...
Page 74: Switching On The System
LSM 510 DuoScan Carl Zeiss Switching on the System LSM 510 META DuoScan Switching on the System The LSM system is equipped with a main power switch located at the front of the System Electronic Rack and two further switches labeled System/PC and Components. The main switch has to be set to ON to be able to switch on and off the system using the System/PC and Components switches.
Page 75: Switching On The Enterprise Uv Laser
LSM 5 software program. This function should preferably be performed by authorized service personnel. See printed manual CHAPTER ROUTINE MODE AND TOOLS. LSM 510 icon Start the LSM 5 software program for the LSM 510 DuoScan and LSM 510 META LSM 510 DuoScan laser scanning microscope.
Page 76: Starting The Lsm 5 Program
In that case, it is not necessary that the lasers and the microscope are switched on. • Double-click on the LSM 510 icon on the desktop of WINDOWS to start the LSM 5 software program (Fig. 4-3).
Page 77 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Switching on the System Carl Zeiss Clicking on this button activates the complete LSM hardware (on- Scan New Images line mode). Please note that the Scan New Images button must be activated before setting up the Routine Mode or the Expert Mode.
Page 78: Main Menu
However, since the LSM 5 software is operated more conveniently with the help of the toolbars, only this method of function activation will be described in the following. • Click on the Start Expert Mode button in the LSM 510 Switchboard window. − The LSM 510 - Expert Mode Main menu appears on the screen.
Page 79 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Main Menu Carl Zeiss Makes it possible for the user to store frequently used processes Macro button (Macro recorder) and to run them automatically (Macro play). It is possible to write new macros or to edit existing ones.
Page 80: File Menu
LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan File Menu The functions of the File menu permit images and the relevant information to be managed and handled completely in a database system. You can also create your own databases. The databases allow images to be stored, loaded and deleted.
Page 81: Create New Image Database
LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss 4.4.1 Create New Image Database The New function permits the creation of a new image database. • Click on the New button in the File subordinate toolbar of the Main menu.
Page 82: Open Existing Image Database
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan 4.4.2 Open Existing Image Database The Open function allows one or several databases to be opened. The images stored in the database(s) are displayed in thumbnail form; they can be selected and loaded into the Image Display window (see page 4-247).
Page 83 LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss Load button Load image and parameter from image database to Image Display window. Load image and parameter with size reduction from image Subset button database to Image Display window.
Page 84: Form Display Mode
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan 4.4.3.1 Form Display Mode When a image database is opened, the Form display mode is used, if no other settings were made under Settings in the Options subordinate toolbar.
Page 85 LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss The currently selected image is displayed as thumbnail in the Image Display window on the right. In the case of Z Stacks or time series, the Slice slider appears on the screen beside the Image Display window.
Page 86: Gallery Display Mode
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan 4.4.3.2 Gallery Display Mode • Click on the Gallery button. All images of the image database, e.g. Convallaria.mdb, (image series) are shown in a tiled arrangement of thumbnails on the screen.
Page 87: Load An Image Into The Image Display Window
LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss 4.4.3.3 Table Display Mode • Click on the Table button. − All images of the image database, e.g. Convallaria.mdb, (image series) are shown in Table display mode on the screen.
Page 88: Load Image With Reduced Resolution (Subset)
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan 4.4.3.5 Load Image with Reduced Resolution (Subset) The Subset function allows images to be loaded with reduced resolution. For this purpose, the image pixels in XY(Z) are reduced. It is also possible to reduce the number of slices (in stacks and time series).
Page 89: Display Images With Certain Features (Filter)
LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss • If the images shall only be copied, also press the Ctrl key during the Drag & Drop procedure (in addition to the rectangular, a "+" sign will also appear near the mouse button).
Page 90 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan The following operator signs can be activated under Operator: equals > larger < smaller >= larger, equals <= smaller, equals <> smaller, larger • Select the relevant operator sign (e.g.: =) from the Operator list box.
Page 91: On Filter Function
LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss If required, the filter features displayed in the work box can be stored. • Click on the Add to List button. The Add to List window will be opened.
Page 92: Save An Image To The Image Database
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan 4.4.4 Save an Image to the Image Database The Save function allows to store an image together with the acquisition parameters (and processing information) to be stored in an image database.
Page 93 LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss • Click on the New MDB button. − This opens the Create New Database window in which you can create a new image database, e. g. Projections.mdb (see Create New Image Database, page 4-19).
Page 94 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan Fig. 4-21 Database window • Click on the OK button in the Save Image and Parameter As window. − The Projections.mdb - AIM window now shows the saved image.
Page 95: Import Of Images
LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss 4.4.5 Import of Images The Import function enables the import of externally created images into the Image Display window and the image database of the LSM 5 software.
Page 96: Export Of Images
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan 4.4.6 Export of Images The Export function allows the export of acquired images and images loaded from the image database. • Select the image to be exported.
Page 97: Multi Print
LSM 510 OPERATION IN EXPERT MODE LSM 510 META File Menu Carl Zeiss 4.4.7 Multi Print This function permits you to arrange several images on one print page and to print them out together. • Click on the Multi Print button in the File subordinate toolbar of the Main menu.
Page 98 LSM 510 DuoScan Carl Zeiss File Menu LSM 510 META DuoScan Closed polyline button: Creation of a closed polyline figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure.
Page 99: Printing Images
• Click on the Exit button in the File subordinate toolbar of the Main menu. − The LSM 510 - Expert Mode Main menu will be closed and the LSM 510 Switchboard menu appears on the screen.
Page 100: Acquire Menu
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Acquire Menu For preparing and acquiring a scanning image, it is recommended to call up and use the tools of the subordinate toolbar in the following order: •...
Page 101: Laser Control
The addition (LSM) indicates the lasers which are provided for imaging and manipulation through the LSM 510 or LSM 510 META scan head. All other lasers are only available for ma- nipulation using the LSM DuoScan.
Page 102: Settings
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.1.3 Settings • Click on the desired laser on the (upper) Lasers panel. − This highlights the selected laser. On the lower panel of the Laser Control window, activate the laser as follows: This applies to the Coherent UV-laser 653 II (Enterprise) and the Ar-multiline laser •...
Page 103: Microscope Control
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss This applies to HeNe and Diode lasers: • After selecting the laser, click on the On button. − The required laser for image acquisition is now available.
Page 104: Microscope Control Window For Axio Imager.z1
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.2.2 Microscope Control Window for Axio Imager.Z1 • Click on the Micro button in the main menu. • The microscope window opens in the last saved configuration.
Page 105 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Recording of microscope settings The upper part of the Microscope Control window shows the recording functionality of microscope configurations. Complete microscope configurations created and applied. The Store button permits existing microscope configurations to be stored under any name.
Page 106 LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Delete a Microscope Configuration A no longer required microscope configuration can be deleted as follows: • Select the microscope configuration to be deleted from the microscope configuration list box.
Page 107 Acquire Menu Carl Zeiss Fig. 4-29 LSM 510 with Axio Imager.Z1 Microscope settings on Axio Imager for transmitted-light observation • Set the reflector turret position to None and click on the On button for transmitted light. • Control the brightness of the halogen lamp with the potentiometer (4-29/4) or the Intensity % slider in the Transmitted Light panel.
Page 108 LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Microscope settings on Axio Imager for reflected-light observation (Epi-fluorescence) • Turn on the HBO 100 W power supply with switch (4-29/1). • Click on the RL reflected light button. The shutter opens.
Page 109: Microscope Control Window For Axiovert 200 M
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Enables the attenuation control via LSM Software. This is also enabled Remote when clicking the ON button. Opens the shutter in the reflected light path and the internal shutter of the lamp.
Page 110 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Recording of microscope settings The upper part of the Microscope Control window shows the recording functionality of microscope configurations. Complete microscope configurations created and applied.
Page 111 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Delete a microscope configuration A no longer required microscope configuration can be deleted as follows: • Select the microscope configuration to be deleted from the microscope configuration list box.
Page 112 J E C F L E Fig. 4-32 LSM 510 with Axiovert 200 M BP Microscope settings on Axiovert for transmitted-light observation • Click on the Reflected Light button and set the shutter to the Closed position. • Click on the Transmitted light button. Click on the On button in the Transmitted Light panel and set the transmitted light intensity via the slider or click on 3200 K.
Page 113: Working In Lsm Mode
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Microscope settings on Axiovert for reflected-light observation (Epi-fluorescence) • Turn on the HBO 100 power supply switch (4-32/1). • Click on the Reflected Light button and set the shutter in the Open position.
Page 114: Configuration Control
8 (incl. monitor diode, transmission and ratio channels), depending on the configuration. The number of channels to be defined in one track for LSM 510 META DuoScan systems is 8 (including monitor diode and transmission detector). Furthermore, 2 ratio channels can be used.
Page 115: Open / Close The Configuration Control Window
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss 4.5.3.1 Open / Close the Configuration Control Window • Click on the Config button in the Acquire subordinate toolbar. − The Configuration Control window is opened with the display last selected.
Page 116: Laserline Button
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.3.3 Laserline Button The Laserline button opens the Wavelength Switch Control window. If more than 8 excitation laser lines (wavelengths) are available from the connected lasers, these can no longer be completely displayed in the Line Active column of the Excitation window.
Page 117 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss • Click on the desired configuration. − The selected configuration is shown in the first line of the Configurations list box (e.g.: FITC/Rhod). • Click on the Apply button.
Page 118: Settings For Single Track In The Channel Mode
The Fiber Out port of the LSM 510 permits connection to another detector. For the configuration of the beam path, please refer to the application-specific configurations depending on the used dyes and markers and the existing instrument configuration (e.g.: module LSM 510 - Config. 26) listed in the annex.
Page 119 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Fig. 4-39 Configuration Control window Beam path - HFT main dichroic beam splitters and NFT secondary dichroic beam splitters • On the Beam Path and Channel Assignment panel, click on the HFT main dichroic beam splitters (see Fig.
Page 120 For channels 1 and 2, it is possible to Fig. 4-40 Configuration Control window change the filters directly on the LSM 510 scan module (see CHAPTER ANNEX: Filter Change in the Detection Beam Path of Channels 1 and 2 of the printed manual).
Page 121 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Further colors for the corresponding channel can be produced as follows: • Clicking on the Define button will open a further Channel Colors window. All the available colors are shown as buttons in the Current Set of Channel Colors panel.
Page 122 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Beam path - Laser attenuation • On the Beam Path and Channel Assignment panel, click on the Excitation button. − This opens a dialog box of all available lasers...
Page 123: Settings For Multi Track In The Channel Mode
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss 4.5.3.6 Settings for Multi Track in the Channel Mode The Multi Track function permit several tracks to be defined as one configuration (Recording Configuration) for the scan procedure, to be stored under any name, reloaded or deleted.
Page 124 LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan The Add Track, Store/Apply Single Track and Remove buttons permit individual tracks to be added, saved or deleted. In addition, this panel is used to activate / deactivate the tracks for the scan procedure.
Page 125 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Settings An additional track is added to the configuration list. The maximum of Add Track button four tracks can be added. One track each with basic configuration is added, i.e.: one Ch 1 channel is activated, all laser lines are switched off,...
Page 126 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Config button in Multi Track mode The Config button in the Multi Track mode permits all tracks to be loaded, stored under any name, or deleted.
Page 127: Ratio Settings Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Delete a Channel Mode configuration A no longer required recording configuration can be deleted as follows: • Click on the Config button, the Recording Configurations window appears on the screen.
Page 128: Settings In The Lambda Mode
4.5.3.8 Settings in the Lambda Mode The Lambda Mode settings are only available if the LSM 510 contains the META detector. With this spectral module the overall emission from the sample is directed onto a wavelength-dispersive element and is imaged on a 32 channel detector.
Page 129 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Control buttons Close Closes the Configuration Control window. Spectra The Spectra button opens the Detection Spectra & Laser Lines window (see page 4-53). Laserline The Laserline button opens the Wavelength Switch Control window (see page 4-54).
Page 130: Settings In The Online Fingerprinting Mode
LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Beam path - META detector settings On the Beam Path and Channel Assignment panel use the two sliders to define the spectral detection range of the META module. The current position of the slider is displayed in a white box when keeping the left mouse button pressed.
Page 131: Non Descanned Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Control buttons for reference spectra Menu for selecting a display color and a reference spectra (reference spectra RS1 ... 8 derive from earlier experiments via mean of ROI or ACE tools); selected spectra and colors appear in the wavelength chart.
Page 132: Camera Detection Panel
Acquire Menu LSM 510 META DuoScan 4.5.3.11 Camera Detection Panel The use of this function permits the use of a Zeiss AxioCam (HRm, HRc MRm) camera as an alternative external detector. • Click on the Config button in the Acquire subordinate toolbar of the main menu.
Page 133: Scan Control
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss 4.5.4 Scan Control The scan parameters for image acquisition are set in the Scan Control window. The microscope must be in the LSM mode, i.e. the LSM button on the tube of the microscope stand...
Page 134: Open / Close The Scan Control Window
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.4.1 Open / Close the Scan Control Window • Click on the Scan button in the Acquire subordinate toolbar of the Main menu. − This opens the Scan Control window, which shows all lasers connected to the system.
Page 135 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Additional button in the Spot mode Scans one image and displays a cross hair which can be freely positioned Spot Sel button within the image window to define the spot for imaging.
Page 136: Frame
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.4.2 Frame When the Frame button is activated, a frame of variable size is scanned pixel by pixel and line by line. The laser beam is moved over the specimen line by line.
Page 137 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Speed panel • Select the Scan Speed from the 13 preset steps via slider or input box. Recommended: 7 for the first scan. A click on the Max button sets the Fig.
Page 138 LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Averaging can be performed in different ways, depending on whether the Mean or Sum method has been activated. If you are using the Mean method, the image information is generated by adding up all scans pixel by pixel and then calculating the mean value.
Page 139 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss − A click on the center button will recenter the scan area to the field of view. − Clicking, holding and drawing the rectangle with the mouse permits the scan area to be moved directly within the field of view.
Page 140 LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan • The sliders (and the relevant arrow buttons) near Detector Gain, Ampl. Offset and Ampl. Gain enable you to set the photomultiplier of the selected channel during continuous scanning.
Page 141 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Excitation panel • In the Excitation panel you can select other lasers and vary laser intensities (in the same way as in the Laser Control or Configuration Control window) and you can program the AOTF for different laser lines.
Page 142 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Fig. 4-60 Image Display window with two tracks plus ratio track (Split xy mode) Fig. 4-61 Image Display window with a Lambda Stack 4-80 B 45-0021 e...
Page 143 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Z Stack This function permits a series of XY-images to be produced in different focus positions (Z slices). When the Z Stack button is pressed, the Z Settings button is automatically activated and the Z Settings panel is displayed in the Scan Control window.
Page 144 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Fig. 4-63 Optical Slice window Tabs Z Sectioning: Tab for setting of Number of Slices, Interval and Current Slice via slider / arrow button. Mark First/Last: Tab for determination of the Z-value for the first and last XY-image of the stack, combined with manual focusing or Stage control.
Page 145 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss The parameters of a Z Stack can be defined using the Z Sectioning tab, the Mark First/Last tab or - if the optional Piezo focus for objectives is connected - the Hyperfine Z Sectioning tab.
Page 146 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan The Line toolbar permits you to define the position, shape, width and color of the cutline in the Image Display window. The following function buttons are available: Arrow selection button: Activates the mouse pointer for the selection and positioning of the cutline in the Image Display window and for changing its length.
Page 147 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Fig. 4-65 Image Display window with cutline displayed • Then click on the Range button. − The XZ-scan will be performed and displayed in the Image Display window. At the same time, the position of the current slice is shown with a green line and the positions of the first and last slice with two red lines.
Page 148 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Fig. 4-66 Scan Control window and Image Display window • Moving the green line (current slice) enables you to change the current focus position (moving the stage or nosepiece in the process).
Page 149 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Mark First/Last tab The determination of the optimum stack size is performed here via focusing during a continuous scan. • Click on the XYcont button. − A continuous XY-scan of the set focus position will be performed.
Page 150 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Hyperfine Z Sectioning tab Activation of this tab is only possible if the piezo objective focusing device has been connected. The piezo objective focusing device can be controlled via software (see Stage, page 4-122).
Page 151 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Auto Z Corr. The function Auto Z Correction allows a linear variation of Detector Gain, Ampl. Offset, and Ampl. Gain values between the different slices of a stack.
Page 152 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. The laser will automatically stop scanning when the Z Stack is completed.
Page 153 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Use ROI (Region Of Interest) Performance of the Frame and Z Stack scan modes can be limited to one or several freely definable sections within the Image Display window using the Use ROI function.
Page 154: Line
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.4.3 Line In the Line mode, fluorescent or reflected light along a freely definable line is displayed in the form of an intensity profile. All the possibilities of creating an image (Frame, Z Stack) are also available in the Line mode.
Page 155 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Fig. 4-73 Image Display window after activation of the Line Sel button Buttons in the Line toolbar The following function buttons are available in the Line toolbar: Arrow selection button: Activates the mouse pointer for the selection and positioning of the scan line in the Image Display window and for changing its length.
Page 156 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Defining a straight line as the scan line • Activate the Line arrow button of the Line toolbar. Click on the spot in the frame at which the line is to start and keep the mouse button pressed.
Page 157 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss If no sufficient coincidence of the two lines can be achieved by the reduction of the scan speed, you have to calibrate the scanner position signal. • Click on the Maintain button in the Main menu and then on the Spline button.
Page 158 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Fig. 4-75 Image Display window after definition of a freehand shape curve for the line scan process Selecting the width and color of the scan line •...
Page 159: Spot
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss 4.5.4.4 Spot In the Spot mode fluorescent or reflected light occurring from a single voxel xyz is detected. In this mode a spot can be defined by two perpendicular lines in the Image Display window.
Page 160: Camera Control
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.4.5 Camera Control The use of this function permits the control of the external CCD-camera settings. Open / Close the Scan control window for camera control •...
Page 161 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Starts a prescan and sets the exposure time automatically. Find In case of a camera multitracking, only one channel should be selected in Configuration Control in order to speed up the find function.
Page 162: Edit Roi (Region Of Interest)
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.5 Edit ROI (Region Of Interest) A scan image allows certain areas (ROIs) to be defined. Only these areas of interest will be scanned. The laser beam will be switched on only in these areas via Acousto-Optical Tunable Filters (AOTF).
Page 163 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss ROI Lists panel In the ROI Lists panel, all the currently defined and stored ROI configurations are shown. • Click on the ROI configuration which you want to use for the scan procedure.
Page 164 Carl Zeiss Acquire Menu LSM 510 META DuoScan Check box (e.g.: 1 - 4): Clicking on this check box allows a ROI to be deactivated. The tick disappears from the check box, as does the relevant marked area from the scanning image.
Page 165 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss • Click on the marking area and keep the mouse button pressed to drag the area into the region of interest in the scanning image. The marking area will be numbered automatically and entered in the Interactive ROI Definition panel with its position and dimension parameters and the appropriate number.
Page 166: Time Series
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.6 Time Series The Time Series Control window allows the definition of parameters for time series. The Time Series function offers the following options for the creation of image series: •...
Page 167: Start Series Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Pause button Interrupts the Time Series. Button labeling is changed to Resume. A current scan is performed until the end. When the button is pressed again, the Time Series is immediately continued with the next scan procedure.
Page 168 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Start via Trigger For the start via trigger control (Trigger button activated), first determine the trigger key which is to trigger the start of the Time Series.
Page 169: Stop Series Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss 4.5.6.3 Stop Series Panel In this panel, the parameters for the end of the time series are set and the number of cycles is determined. Fig. 4-87...
Page 170: Cycle Delay / Time Interval Panels
LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan If the entered number of cycles has been processed without a trigger impulse having been given to end the procedure, the Time Series is finished. If a trigger signal is given before the cycles have been processed, the Time Series will only be interrupted.
Page 171 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Function description List of the stored sets of time delays or Time Intervals for time series. Time delay or Time Interval list box Application of the sets of delays for time series selected in the list box.
Page 172: Marker Panel
LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan • Click on the Delete button. The set will be removed. The Time buttons can also be activated via keys 1 to 4 of the Trigger Control. • Click on the required Time button.
Page 173 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Setting of a marker during the scan procedure. Set 1-7 button Description Entry of the comments for the marker. input box (1-7) Selection of the trigger key (1-4) with which the marker is to be set.
Page 174: Time Series Of A Frame
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.6.6 Time Series of a Frame • Set the relevant parameters for time control in the Start Series, End Series and Time Delay panels. • Start the Time Series with a click on the Start T or Start B button.
Page 175 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss Fig. 4-93 Image Display window of a Time Series Scan The image markers have different colors with the following meaning: • red: manually set marker with time indication and comments •...
Page 176: Time Series Of A Frame Over Z Stack
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.6.7 Time Series of a Frame over Z Stack • First, set all parameters required for recording a Z Stack in the Scan Control window.
Page 177: Time Series Of A Frame Over A Z Stack Over Lambda
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss For Z Stacks over the time (4D) following offline functions will be enlarged: − Slice (Z slider and Time slider) − Gallery (Z, Time and Z + Time buttons) −...
Page 178: Time Series With Mean Roi
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.6.9 Time Series with Mean ROI • Set all the parameters in the same way as for Time Series of a frame. • Then click on the Mean ROI button in the time series frame.
Page 179 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss The following functions are available: Display of the data of the ROIs used for the creation of the MeanROI (identical to the Edit ROI window). If the check box of a ROI is deactivated, the ROI's intensity values are no longer displayed in the Intensity-Time diagram.
Page 180: Edit Bleach
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan 4.5.7 Edit Bleach The use of this function permits the setting of bleaching parameters for spot, line or frame bleaching. 4.5.7.1 Open / Close the Edit Bleach Window •...
Page 181: Settings Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss 4.5.7.2 Settings Panel The Settings panel allows you to determine when, where and how the bleaching process shall be done (only works in connection with time series).
Page 182: Bleach Parameter Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan If this check box is ticked , you can set a different XY position for spot Different XY Spot Bleach Position: bleaching. This function is only available using the Spot scanning mode. Click on the Spot Select button in the Scan Control window.
Page 183: Excitation Of Bleach Track Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss • Enter the number of iterations of the bleaching procedure in the Iterations input box. • Click on the Define Region button. − The Bleach Regions window appears.
Page 184: Start / End A Bleaching Procedure
Z and X, Y positions. By use of an LSM 510 META scanhead on an Axiovert 200 M sideport system care should be taken when moving the...
Page 185: Open / Close The Stage And Focus Control Window
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss 4.5.8.1 Open / Close the Stage and Focus Control Window • Click on the Stage button in the Acquire subordinate toolbar of the Main menu. − The Stage and Focus Control window appears on the screen.
Page 186: Stage Position Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan Focus Wheel check box Clicking on this check box activates / deactivates the focus wheel of the microscope. Use of the optional piezo objective focusing device The HRZ Step slider is used to set the step width of the fine focusing device.
Page 187 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss If Manual is active, the scanning stage can be moved manually via the knurled screws. The Move To and Center function buttons in Stage Position are without a function. The Current Position is updated.
Page 188: Tile Scan Panel
LSM 510 DuoScan Carl Zeiss Acquire Menu LSM 510 META DuoScan HRZ Zero button 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.
Page 189 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Acquire Menu Carl Zeiss • Click on Start. − The overview frame is scanned and displayed on the screen in a new Image Display window. Fig. 4-106 Image Display window of a Tile Scan •...
Page 190: Vis, Tv And Lsm Buttons
(if connected) via camera adapter of the binocular tube − LSM: screen observation via laser excitation using the LSM 510 and software evaluation If the beam path of the microscope is changed manually via buttons on the tube this is recorded by the software and the relevant button is activated automatically.
Page 191: Process Menu
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss Process Menu The functions of the Process menu permit already stored scan images to be subsequently linked and processed using mathematical functions and algorithms. • In the Main menu toolbar, click on Process.
Page 192: Source Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.1.2 Source Panel In the Source 1 panel, the first image source for the addition process is determined. The current image is displayed in the display box of the image selection box.
Page 193: Add Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss If an existing Image Display window is used to perform the Add function, you must determine whether an existing channel shall be overwritten with the Add operation or whether a new channel shall be added.
Page 194: Subtract
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.2 Subtract The Subtract function links two channels each of one or two images into a new channel by subtraction. The channel created in this way can be stored via the Save As function.
Page 195: Multiply
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.3 Multiply The Multiply function permits two channels each to be linked into a new channel by multiplication. The channel created in this way can be stored via the Save As function.
Page 196: Ratio
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.4 Ratio The Ratio function permits to create a new image or image series by offsetting two images or image series against each other. The channel created in this way can be stored via the Save As function.
Page 197: Formula Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.4.3 Formula Panel The formulas can be typed in using the keyboard. • Click the Operators button to open the permissible operators list. • Select the operator(s) from this list (Fig. 4-116) and click the Insert button.
Page 198: Duplication (Image)
LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan − The image of the copied channel is then displayed in a new window or in the Image Display window activated for it. • The new image can be stored via the Save As function.
Page 199: Filter List And Edit Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss Proceed as follows to select an image via the image selection box: • Click on the arrow button. The image selection box is opened and all the currently loaded images are displayed in a minimized form.
Page 200 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan Sharpness filter With the sharpness filter, the original image is filtered with a lowpass filter first. The result of this filtering is then subtracted from the original image.
Page 201 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss User-defined filter The User-defined function permits you to create your own filters. In addition to the Kernel Size, the parameters Factor, Divisor and Offset can be modified here.
Page 202: Preview Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.7.4 Preview Panel The Preview function allows you to have the result of the Filter operation displayed as a preview image. Fig. 4-124 Preview panel •...
Page 203: Interpolate
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.9 Interpolate This function permits the continuous contrast and brightness change in a Z- stack or Z- stacks over time through interpolation between the starting and end values. This permits the subsequent...
Page 204: Interpolation Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.9.3 Interpolation Panel In the Interpolation panel, the parameters for the interpolation procedure are set. • Use the Start Image slider to select the slice at which the interpolation procedure shall start.
Page 205: Preview Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.9.4 Preview Panel The Preview function enables you to see the result of interpolation for one slice each in a preview window. Fig. 4-128 Preview panel • Activate the Preview check box with a click of the mouse.
Page 206: Image Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.10.2 Image Panel • Click on the arrow button. The image selection box will be opened and all the currently loaded images are displayed in a minimized form.
Page 207: Preview Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.10.4 Preview Panel • If Preview is activated, a preview of the shift is shown in a separate Image Display window. Fig. 4-132 Preview panel 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.
Page 208: Unmix
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.11 Unmix The Unmix functionality permits to extract the emission of single fluorescence dyes (e.g. GFP only, YFP only etc.) from the overall emission band of strongly overlapping multifluorescence signal intensities by a pixelwise linear unmixing procedure .
Page 209 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss Generate channels with Residuals shows the difference between the offered spectrum and the resulting image in intensity values. The higher the intensity in this additional channel the worse the fit of the spectra to the dataset.
Page 210 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan Fig. 4-135 Image Display window before unmixing Fig. 4-136 Image Display window after unmixing 4-148 B 45-0021 e 03/06...
Page 211: Ion Concentration
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.12 Ion Concentration The use of this function (option) permits the calibration of ion concentrations physiological experiments. 4.6.12.1 Open / Close the Ion Concentration Window • Click on the Ion Conc button in the Process subordinate toolbar of the main menu.
Page 212: Single Wavelength Dyes - Offline Calibration
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.12.3 Single Wavelength Dyes – Offline Calibration • Subtract background/autofluorescence image from raw images to obtain. • Perform equation- or titration calibration (compare F with a calibration curve → titration calibration or put F values in calibration formula).
Page 213: Ratiometric Dyes
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.12.4 Ratiometric Dyes • Fura-2, Indo-, SNARF, Cameleon, Ratiometric Pericam, Phluorin,... • Display fluorescence ratio R over time • Display fluorescence ratio R corrected for background/autofluorescence over time •...
Page 214: Ratiometric Dyes - Calibration
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.12.6 Ratiometric Dyes - Calibration • Subtract background/autofluorescence images from raw images to obtain )/(F korr 1Background 2Background when calibration reference is not obtained with the experimental sample (in situ) •...
Page 215: Ratiometric Dyes - Equation Calibration (Grynkiewicz)
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.12.7 Ratiometric Dyes - Equation Calibration (Grynkiewicz) Fura-2, Indo-1,.. (dissociation constant) taken from literature : derived from ion-free state of the dye (e.g. 0 R max: derived from ion-bound state of the dye (e.g.
Page 216: Function Description
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan 4.6.13.2 Function Description Click into window Allows to select the image series of the FRAP experiment to be analyzed by clicking into the appropriate image window.
Page 217: Example: Frap Performed In A Nucleus Expressing Gfp Labeled Proteins
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss 4.6.13.3 Example: FRAP Performed in a Nucleus Expressing GFP Labeled Proteins Display of the image series in the Mean ROI display mode: The drawing tools are used to define the ROI to be analyzed (ROI 1), the background ROI (ROI 2), and the reference ROI (ROI 3).
Page 218 LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan − The amplitude of the fitted curve (which equals the mobile fraction) I1 mobile fraction. − The fitted parameter T1 (seconds). − The rate constant for the exchange of molecules between the bleached region and the surrounding area K (per second).
Page 219 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Process Menu Carl Zeiss If the analysis is done using the double exponential fit the fitted curve displays the mean of the fitted values for the two different mobile fractions. The table shows the following additional parameters: −...
Page 220: Image Scaling
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Process Menu LSM 510 META DuoScan Fig. 4-145 Schematic FRAP curve with marks at the relevant data points. I0, I1 and I delta which are calculated performing the Kinetic Analysis 4.6.14 Image Scaling The use of this function permits the scaling of a imported image in voxel dimensions.
Page 221: View Menu
LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss 3D View Menu The 3D View functions serve to record and play back series of images for 3D display of microscopic structures. • In the Main menu toolbar, click on 3D View.
Page 222: Source Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss 3D View Menu LSM 510 META DuoScan 4.7.1.2 Source Panel In the Source panel, the image source is selected. The currently selected image is displayed in the display box of the image selection box. Proceed as follows to select an image via the image selection box: •...
Page 223 LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss Transparency tab The color is defined by the z position of the brightness value. Mode Maximum: The transparent projection is built up from the rear to the front. The color is...
Page 224: Preview Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss 3D View Menu LSM 510 META DuoScan 4.7.1.4 Preview Panel The Preview function permits you to regard the influence of parameter changes in an Image Display window. • After finding the optimum adjustment using the...
Page 225: Projection Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss 4.7.2.3 Projection Panel • On the Projection panel, set the parameters needed for the animation: Turning Axis, First Angle, Number Projections and Difference Angle in the Projection tab and the Mode parameters in the Transparency tab.
Page 226: Preview Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss 3D View Menu LSM 510 META DuoScan Transparency tab The color is defined by the z position of the brightness value. Mode Maximum: The transparent projection is built up from the rear to the front. The color is...
Page 227 LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss • After finding the optimum adjustment using the Preview function, you have to generate the final version of the image using the Apply button. − The projection appears. The computation can be followed in the image or by the progress bar.
Page 228: Stereo
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss 3D View Menu LSM 510 META DuoScan 4.7.3 Stereo Stereoscopic images can be generated in a variety of ways by means of the Stereo function. A stack of images must be available.
Page 229: Stereo Images Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss 4.7.3.3 Stereo Images Panel • In the Stereo Images panel, set the parameters needed for stereoscopic viewing: Mode, Basic Angle, Right Left Angle, Number Images and Difference Angle in the Projection tab and the Mode parameters in the Transparency tab.
Page 230 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss 3D View Menu LSM 510 META DuoScan Transparency tab The color is defined by the z position of the brightness value. Mode Maximum: The transparent projection is built up from the rear to the front. The color is...
Page 231: Preview Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss 4.7.3.4 Preview Panel The Preview function permits you to regard the influence of parameter changes in an Image Fig. 4-160 Preview panel Display window. The Slice slider enables you to select the slice which shall be displayed in the Preview Image Display window.
Page 232: Deconvolution (Dcv)
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss 3D View Menu LSM 510 META DuoScan 4.7.4 3D DeConVolution (DCV) The 3D Deconvolution option is used for the resolution enhancement of fluorescence image stacks. When a three-dimensional object is reproduced by an optical system the resulting image of the object does not correspond exactly to the object's actual form.
Page 233: Open / Close The 3D Deconvolution Window
LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss As the resolution of an optical system is significantly lower in the axial direction than in the lateral (X/Y-)direction, the greatest improvement in resolution can be achieved in the Z-direction.
Page 234: Source Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss 3D View Menu LSM 510 META DuoScan 4.7.4.2 Source Panel The image to be processed is selected in the Source panel. The currently selected image is shown in the display box of the image selection box.
Page 235 LSM 510 OPERATION IN EXPERT MODE LSM 510 META 3D View Menu Carl Zeiss Activation of the Auto detect check box will start a routine for the automatic determination of the noise level in the entire image part of the Z Stack (not available in the Nearest Neighbour method). If Auto detect is enabled, the Restoration Effect slider is disabled.
Page 236: Macro Menu
Macro language in the "Microsoft Word for Windows" and "Microsoft Excel for Windows" products. Experience with "Microsoft Visual Basic" would also be beneficial for macro-programming of the LSM 510. An Integrated Development Environment, called IDE in the following, is available for the editing and debugging of macros.
Page 237: Macro Control
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss 4.8.2 Macro Control 4.8.2.1 Open / Close the Macro Control Window • Click on the Macro button in the Macro subordinate toolbar of the Main menu. − This opens the Macro Control window.
Page 238 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Fig. 4-172 Macro panel Macros are stored and managed in project files (*.lvb). Before you can record or edit a macro, you have to create a project as follows: •...
Page 239 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Recording panel Before recording a command sequence, you can enter the name for the macro to be created in the Rec Name input box of the Recording panel.
Page 240: Assign Macro To Button Function
LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Proceed as follows to perform a macro: • Select the required macro from the Macros list box of the Recording panel. • Click on the Run button to start performing the macro.
Page 241: Editing And Debugging Of Macros
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Delete the linking of a button in the Macro subordinate toolbar Proceed as follows to delete the linking between a button in the Macro subordinate toolbar and a macro: •...
Page 242: Overview Of Available Macros
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan 4.8.3 Overview of Available Macros Documentation files (*.rtf, *.doc) of advanced macros will be located in the macro directory. Name Description AOTFfitlin.lvb Linearize laser attenuation (AOTF or mechanical) Autofocus.lvb...
Page 243 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Name Description LsmTime.lvb Triggered Time scan Macro MetaExport.lvb Export of META image files including all channels as tif, bmp, ... ModifySeries.lvb Modifies Z Stacks and Time Stacks like Rotation of the stacks, being mirrored, Conversion of time stacks into z-stacks and vice versa;...
Page 244: Sample Macros
ScanfieldTransform Macro The Macro enables to match the scan fields of the LSM 510 or LSM 510 META with the LSM DuoScan. The procedure is necessary to make sure that the LSM DuoScan can also be used for bleaching, activation or uncaging of a selected ROI.
Page 245 Sample Configuration for imaging the • Tick the checkbox for the Test grid. test slide in reflection using the Argon Laser of the LSM 510 • Set up a track for reflection imaging with the scanner chosen in the Macro.
Page 246: Distance Macro - Distance.lvb
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Fig. 4-179 Projection of the Test grid and the scanned image of the test slide into the image window Red represents the projection of the Test grid. Green represents the scanned image of the test slide.
Page 247: Profile Macro - Profile.lvb
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss 4.8.4.3 Profile Macro – Profile.lvb This macro permits the gray values of a line created in the scanning image to be determined pixel by pixel. • Click on the Profile button in the Macro subordinate toolbar.
Page 248: Excitation Fingerprinting
3.2 or higher. With the LSM 510 NLO and LSM 510 META NLO in combination with a software tunable ultra fast laser (multiphoton lasers such as Chameleon from Coherent or Mai Tai from Spectra Physics) one can acquire excitation spectra and use these spectra to perform linear unmixing.
Page 249 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss • Adjust the AOM Transmission to keep the power indicated at the power meter constant, then press Add and + 10 steps again. • Repeat this procedure until you have acquired the AOM settings for all wavelengths; 10 nm steps are sufficient.
Page 250 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Fig. 4-182 Example using a Chameleon Laser If the laser does not mode lock after several minutes press Recovery and wait for mode locking (takes three to five minutes). Do not tune to a new wavelength if the laser status is indicated as busy! If the tuning has been done during the status “busy”...
Page 251 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss − Advanced Linear Unmixing: The algorithm used for Linear Unmixing calculates the minimal difference between the unknown spectra and the reference spectra for all possible mixtures of the reference spectra (lowest square fit of acquired spectrum).
Page 252: Concatenate Images
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan 4.8.4.6 Concatenate Images System requirements: − LSM 5 system, software release 3.5 or higher This program combines open image files (single images, z-stacks, time series) to a single file series.
Page 253 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Examples: (1) Concatenate two images: • Open Concatenate Images macro • Click Add button followed by a click in the open image window. An image link will appear in the row of icons below (time line).
Page 254: Multiple Time Series Macro
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan 4.8.4.7 Multiple Time Series Macro The Multi Time Series program is designed to provide flexible programming of automated time dependent experiments. − A pre-program time delay −...
Page 255: Stitchart Plus Macro
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss 4.8.4.8 StitchArt plus Macro Purpose of the StitchArt plus Macro The StitchArt plus Macro is useful for the acquisition of larger profiles and larger fields of view than a single scan field could cover.
Page 256 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Loading the StitchArt plus Macro. • Click on the Macro button 1. • Click on the macro button 2. − The Macro Control window opens.
Page 257 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Orientate panel Find Focus button The objective chosen in the Multiple Profiles Parameters... window is performed and a fast line scan over Z is acquired. The Z range of this line scan is 0.5 mm, the single Z step 300 nm.
Page 258 LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Select subprofiles from MULTIPLE PROFILE data After acquisition of a Multiple Profile it is displayed as an XZ projection. By variation of the Z:XY ratio slider on the right hand side of the image window the Z:X ratio could be changed.
Page 259 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Setting Parameters for Profile & Stack Stitching • Click on the Parameters ... Button in the Profile & Stack Stitching window. − The Parameters window opens. Pinhole diameter and receiver offset are applied as defined in the Channels tab of the Scan Control window of the LSM 5 software.
Page 260 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Multiple Profile panel In the Multiple Profile panel the parameters necessary for the Multiple Profile procedure can be defined. Objective An objective has to be chosen from the currently used objectives of the stand.
Page 261 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss The X pixel resolution of a slice of a single stack can be varied between 4 and Pixels X 512 Pixels. The Y pixel resolution of a slice of a single stack can be varied between 4 and Pixels Y 512 Pixels.
Page 262 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Calibrating Routine • When clicking on the Calibrate button in the Profile & Stack Stitching window the Calibrate... window opens. Fig. 4-190 Profile & Stack Stitching window...
Page 263 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Saving the images The acquired Multiple Profiles and intensity based Multiple Stacks can either be saved as image stack based data or as a topography based data.
Page 264 LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan The other sections contain offsets for the individual tiles. The section name has the format [Tile(xxx,yyy)] where xxx is the zero based horizontal index of the tile and yyy is the zero based vertical index of the tile.
Page 265: Visual Macro Editor
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss 4.8.5 Visual Macro Editor The Visual Macro Editor is an optional software module which allows the user to program a variety of scanning procedures and image calculations as well as the combination of both.
Page 266: Function Description
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan 4.8.5.2 Function Description The Editor uses predefined action blocks. The action blocks for setting up the work flow of the Macro are categorized according to their properties. The following categories are displayed on the left-hand side of the Visual Macro Editor control window.
Page 267 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss It is possible to define variables. A variable has a (property) name and a mathematical value. The Assignment action block can be used to assign a new value to a variable when inserted into the program flow using mathematical algorithms.
Page 268 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan Acquisition Action Blocks Loads a Beam Path Configuration form the Configuration list. Use the pull down menu in the Property/Value list to select the Configuration that should be used for imaging.
Page 269 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Windows Action Blocks This block has to be used if the actually scanned image(s) should be displayed. For each Image Display block a new image window is opened. If not saved, the image will be overwritten when the program flow is passing by again.
Page 270 LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan This block introduces a delay in the program flow. The delay time is started when the block is reached within the program flow. The block allows to assign a potential error message with a textbox and to decide on the Error where to direct the program flow.
Page 271: Editing A Macro
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss Hardware Control Action Blocks If a motorized stage is available the movement of the stage can be set up as part of the program flow. The stage can be moved to an absolute position which can also be taken from the actual stage position using the Read back function or typed in individually.
Page 272 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Macro Menu LSM 510 META DuoScan • Click the single action blocks and assign the values to the listed properties (beam path and scan parameters can be chosen according to the sample). Fig. 4-194...
Page 273 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Macro Menu Carl Zeiss • Now the first complete scan has been performed and the image is stored. For the next cycle the variable Index has to get another value to make sure the numbering of the images works.
Page 274: Options Menu
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Options Menu LSM 510 META DuoScan Options Menu The Options menu permits performance of the following functions: − User-Defined Examination Methods of the Expert Mode are made available to the Routine Mode.
Page 275: Load Rm Function
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Options Menu Carl Zeiss • Click on the Options button of the Main menu and then on Export RM (see Fig. 4-202). − The Export to Routine Mode window appears. • Enter any name for the method to be taken over.
Page 276 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Options Menu LSM 510 META DuoScan Fig. 4-203 Routine Mode Database window 4-214 B 45-0021 e 03/06...
Page 277: Dye Db Function
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Options Menu Carl Zeiss 4.9.3 Dye DB Function The Dye DB function is for information only and permits access to the database contained in the system, including a list of suitable dyes for fluorescence microscopy.
Page 278: Spectra Db Function
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Options Menu LSM 510 META DuoScan 4.9.4 Spectra DB Function Spectrums created with the Lambda Mode can be stored in the Spectra Database. • Click on the Spectra DB button in the Options subordinate toolbar.
Page 279: Open / Close The Settings For User
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Options Menu Carl Zeiss 4.9.5.1 Open / Close the Settings for user : ... Window • Click on the Settings button in the Options subordinate toolbar of the Main menu. − This opens the Settings for user : ... window.
Page 280: Database General
LSM 510 DuoScan Carl Zeiss Options Menu LSM 510 META DuoScan Export to Attofluor format On activation of this option, newly recorded or modified images are stored by Save in the Attofluor format. The displayed Experimental directory selection box permits selection of the directory in which the data will be stored.
Page 281: Database Table Viewer
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Options Menu Carl Zeiss Use separate path for "Create" and "Open" This option permits the path to be changed when the Open or New database function is used. Save most recently used path at exit and reuse at next program start On activation of this option, the path setting last used is automatically selected again in the Open Database or Create New Database window.
Page 282: Import / Export
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Options Menu LSM 510 META DuoScan 4.9.5.6 Import / Export Use separate path for "Import" or "Export" This option permits the change of the path setting for use of the Import or Export function (File menu).
Page 283: Print Status Display
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Options Menu Carl Zeiss 4.9.5.9 Print Status Display This tab permits selection of which information is displayed in print preview. On activation of the Print Status Information check box, the status information will be printed.
Page 284: Scan Mean Of Rois
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Options Menu LSM 510 META DuoScan 4.9.5.12 Scan Mean of ROIs The Mean of ROIs tab permits the presetting of the Image Display window for the optional MeanROI function (time series) to be changed with regard to scaling and display mode of the intensity time diagrams.
Page 285: Temporary Files
Expert Mode is started. On activation of the Don't show logo check box, the initial screen with the Zeiss logo will not be displayed after the start of the LSM 5 software. Fig. 4-219 Program Start tab 4.9.5.15...
Page 286: Image Display
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Options Menu LSM 510 META DuoScan 4.9.5.16 Image Display The Image Display tab enables you to determine the window toolbars which shall be automatically displayed when an Image Display window is opened.
Page 287: Save
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Options Menu Carl Zeiss 4.9.5.17 Save The Save tab permits the presetting for the storage of scanned or processed images to be changed. Activation of one of the three option buttons...
Page 288: Maintain Menu
The Maintain menu contains additional modules to check and guarantee the interference-free operation of all the software and hardware components of the LSM 510. The LSM 510 and LSM 510 META can be equipped with an additional scanner, the LSM DuoScan. For optimal results the scan fields of the two scanners must be aligned.
Page 289: Optical Calibration With Speed 11-13 (Bidirectional)
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss Calibration conditions Before the calibration process can be started, the system has to be in operation for at least one hour. Calibration procedure • Click on the Scanner button in the Maintain subordinate toolbar of the Main menu.
Page 290 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Maintain Menu LSM 510 META DuoScan Calibration conditions • The microscope stand has to be placed totally vibration-free. − Note, that even power units on the granite plate or inappropriate situated cables can cause vibrations.
Page 291 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss • The calibration standard has to be positioned as indicated in Fig. 4-226. • Before starting the calibration procedure, change to the bidirectional scan mode in the Scan Control window.
Page 292 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Maintain Menu LSM 510 META DuoScan • With activated Auto Calibration box, all speeds (index) and axis are calibrated one after another automatically. Before the optical calibration is done a full calibration must have been performed on the system.
Page 293 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss • If necessary slightly reposition the sample and click on the Next button. • Do not focus or change scan parameters during calibration procedure! • After calibration of X-axis the orientation of the calibration grid in the calibration window changes from horizontal to vertical orientation.
Page 294: Important Notes And Hints
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Maintain Menu LSM 510 META DuoScan 4.10.1.3 Important Notes and Hints The tuning procedure runs automatically to a large extent without any problems. However, several errors can occur. That’s why it is strongly recommended to observe the complete calibration process.
Page 295: Scanner Calibration In Lsm 5 Software
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss Non-regularities of the scanner feedback. The ticks on the outer sides of the grid vary about more than 1 tick width between consecutive images (in the middle of the calibration process, the linearity is optimized and the problem starts to occur).
Page 296: Objective
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Maintain Menu LSM 510 META DuoScan 4.10.2 Objective This function permits changed objectives to be activated and the parfocality to be set without having to exit the software. 4.10.2.1 Objective Change •...
Page 297 The new objective is stored in the database and included in the Change Objectives window. You can now activate the new objective as a favorite objective using the procedure described above. If you have activated the Non Zeiss check box, objectives...
Page 298: Focus Speed Change
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Maintain Menu LSM 510 META DuoScan 4.10.2.2 Focus Speed Change • Change the required objective in the nosepiece. • Click on the Objective button in the Maintain subordinate toolbar of the main menu.
Page 299: Pinhole Adjustment
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss • Click on the Close button to exit the Objective Control window and accept the settings. Fig. 4-236 Objective Control window 4.10.3 Pinhole Adjustment In the Pinhole and Collimator window, the pinholes and collimators are optimally aligned and adjusted to the used beam path (configuration).
Page 300: Pinhole Panel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Maintain Menu LSM 510 META DuoScan 4.10.3.2 Pinhole Panel No further software function can be activated and executed during pinhole adjustment. Pinhole / Description Selection of pinholes (PH1 to PH4) to be adjusted via the Pinhole selection field: box, display of the relevant channel in the Description field.
Page 301: Collimator Panel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss 4.10.3.3 Collimator Panel Collimator Description Selection of the collimator (IR / VIS or UV / VIS) to be adjusted via Collimator field: selection box, display of selected collimator in the Description field.
Page 302: Pinhole And Collimator Adjustment
LSM 510 META DuoScan 4.10.3.4 Pinhole and Collimator Adjustment Adjustment of the LSM 510 pinholes can be performed manually or automatically. If several channels are used to produce the image, all the used pinholes must be adjusted separately. Manual pinhole and collimator adjustment The position of the pinhole relative to the detector in terms of X-Y-Z coordinates contributes substantially to image optimization.
Page 303 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss Automatic pinhole and collimator adjustment The automatic adjustment allows the LSM 510 pinholes to be used with any combination of beam splitters. • Click on the Adjust Automatically button. − The Requirements Adjustment window will then appear.
Page 304: Set Find
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Maintain Menu LSM 510 META DuoScan 4.10.4 Set Find This function permits the preset parameters of the Find function (see Scan Control, page 4-71) to be matched individually. • Click on the Set Find button in the Maintain subordinate toolbar of the Main menu.
Page 305: Camera
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Maintain Menu Carl Zeiss 4.10.6 Camera In the Camera Color Adjustment window the user can adjust the white balance and color balance of a connected camera. Clicking the Pic button allows to set the white balance using the mouse cursor in the camera image.
Page 306: Window Menu
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Window Menu LSM 510 META DuoScan 4.11 Window Menu The Window menu includes the additional functions Full Screen, Close All Image Windows, Toolbar and Scan Information which are not available from a toolbar.
Page 307: Scan And System Information
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Window Menu Carl Zeiss 4.11.3 Scan and System Information This function opens the Scan Information window, in which the current scan data are displayed. The extent of the data displayed in the Scan and...
Page 308: Help Menu
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Help Menu LSM 510 META DuoScan 4.12 Help Menu The Help menu permits activation of the Help function and of a window containing information on the installed software version. 4.12.1 Help •...
Page 309: Display And Analysis Of Images
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13 Display and Analysis of Images 4.13.1 Structure of the Image Display Window The Image Display window shows the image or images when they are −...
Page 310: Display Modes
LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan − line time series (time series of lines and time series with Z Stack of lines) − point time series (time series of points) − Lambda series (Lambda Stack of images and time series with Lambda Stack of images) 4.13.2...
Page 311: Select - Chan
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss All display functions are exclusive functions. Only one can be active at a given time. To generate different views of the same image set use the Duplicate function in the Process menu.
Page 312: Assigning Another Color To A Channel
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.3.1 Assigning Another Color to a Channel • Click on one of the channels button in the Fig. 4-249 Color selection box Channels toolbar (e.g.: Ch1).
Page 313: Select - Zoom
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.4 Select - Zoom This function allows to change the zoom factor of an image displayed. Click on Zoom will display the Zoom toolbar. Any changes done with this toolbar are effective immediately.
Page 314: Select - Slice
LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan When active it allows you to zoom all images of a gallery to the same extent without changing the display image format. Zoom-+, Zoom-–, Zoom 1:1, Zoom-Mouse and All can only be defined when the Zoom-Auto function is deactivated.
Page 315: Select - Overlay
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.6 Select - Overlay This function allows to − select from a set of drawing functions such as rectangles and arrows − add a scale bar to the image −...
Page 316: Buttons In The Overlay Toolbar
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.6.2 Buttons in the Overlay Toolbar The following functions can be used on activation of the buttons in the Overlay toolbar: Arrow (selection) button: Activation of the mouse button for selection, resizing or movement of an overlay element in the Image Display window.
Page 317 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Circle button: Creation of a circle in the Image Display window. Clicking and holding down the mouse button sets the center point, drag the diameter and release the mouse button to end the procedure.
Page 318 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Off button: Deactivation of the display of overlay elements in the Image Display window (hide overlay). Deactivation of the overlay functions. Line button: This button allows you to determine the line thickness of the area outline.
Page 319: Select - Contr
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.7 Select - Contr This function allows to − change the contrast and brightness of an image − change the contrast and brightness of a channel of an image −...
Page 320: Ramp
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.7.1 Ramp The intensity is set via two knots in the Intensity Screen, which allows an intensity line to be created in the form of a ramp.
Page 321: Spline
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.7.3 Spline The intensity is set in the Intensity Screen via a freely selectable number of knots, which permits the creation of an intensity line in the form of a spline.
Page 322: Select - Palette
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.8 Select - Palette This function allows to − change the palette used for displaying the image(s) − define and save new palettes −...
Page 323: Editing And Storing A Palette
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Fig. 4-260 Image Display window, Select - Palette; Color Palette and Add Palette to List window 4.13.8.1 Editing and Storing a Palette A palette is edited by moving the knots in the...
Page 324: Delete A Palette
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.8.2 Delete a Palette Proceed as follows to delete a palette: • Click on the name of the palette to be deleted in the Color Palette List panel and then on the Remove button.
Page 325 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss The animation is controlled via the following function elements: Current Slice slider: Manual movement through the individual slices of a stack by moving the slider, or by entering the slice number in the input box. Slider can be accessed only, when the automatic animation is off.
Page 326: Select - Reuse
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.10 Select - Reuse This function allows to − transfer acquisition parameters of an image from the image data base to the Microscope control, Configuration Control, Scan Control, Time Series Control and Bleach Control windows and applies those parameters directly on the system.
Page 327: Select - Crop
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.11 Select - Crop This function allows to − interactively define the size and orientation of a rectangular scan area on the image displayed in the Image Display window.
Page 328: Select - Copy
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.12 Select - Copy This function allows to − copy the current displayed image into the clipboard. Click on Copy will be immediately effective.
Page 329: Select - Save As
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.14 Select - Save As This function allows to − save the image(s) of the Image Display window into an Image Database − by showing a dialogue −...
Page 330: Display - Split Xy
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.16 Display - Split xy This function allows to − display the individual channels of a multi channel image as well as the superimposed image The settings of Chan, Zoom, Slice, Overlay, Contr and Palette apply.
Page 331: Display - Ortho
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.17 Display - Ortho This function allows to − display a Z Stack of images in an orthogonal view − measure distances in three dimensions −...
Page 332: 4.13.17.1 Ortho - Select Function
LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.17.1 Ortho - Select Function • By changing the parameters X, Y and Z in the Orthogonal Sections toolbar, the section plane can be positioned at any XYZ coordinate of the Z Stack.
Page 333: 4.13.17.2 Ortho - Distance Function
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.17.2 Ortho - Distance Function • Activating the Dist. button permits length measurements in 3D space. • Click on the Mark button to set the first XYZ-point for the measurement of the spatial distance.
Page 334: 4.13.17.3 Ortho - 2D Deconvolution Function
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.17.3 Ortho - 2D DeConVolution Function The 2D deconvolution function causes orthogonal projection enhancement through the computed correction of the resolution in the Z-coordinate.
Page 335: Display - Cut
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss PSF tab (optional with 3D DCV) The objective data are displayed in the PSF tab. In the case of wavelengths above 700 nm, the NLO button is automatically enabled.
Page 336: Display - Gallery
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.19 Display - Gallery This function allows to − display images (Z Stack, time series, combination of both) side by side in tiled fashion −...
Page 337 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Fig. 4-271 Image Display window, Gallery display 03/06 B 45-0021 e 4-275...
Page 338: Display - Histo
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.20 Display - Histo 4.13.20.1 Display - Histo - Overview This function allows to − display a histogram (distribution of pixel intensities) of an image −...
Page 339 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Histogram functions Ignore black pixels (gray value 0) in the histogram. Skip Black button Ignore white pixels (gray value 255 or 4096) in the histogram.
Page 340: 4.13.20.2 Area Function
LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.20.2 Area Function • Click on the Area button in the Histogram toolbar. − The function elements for Area measurement are displayed at the bottom right of the Histogram toolbar.
Page 341 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Closed polyline button: Creation of a polyline figure in the image. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure.
Page 342: 4.13.20.3 Colocalisation Function
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan • Specify the thresholds either with the Threshold low and Threshold high sliders, or with the ▲▼ buttons. 4.13.20.3 Colocalisation Function The Colocalisation function permits interactive analysis of two channels of an image by computing a scatter diagram (colocalisation).
Page 343 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Function elements The following function elements are available: Displays the scatter Histogram of the two image channels Adds display of the according data table...
Page 344 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Enhanced colocalisation Scatter diagram image display and data table are interactively linked. Fig. 4-275 Image Display window, Colocalization display Fig. 4-276 Scatter diagram and threshold with crosshair...
Page 345 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Quantitative Parameters − No. of pixels in image ROI or scatter region − Area / relative area of image ROI or scatter region − Mean intensities / SD within image ROI or scatter region −...
Page 346: Display - Profile
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Overlap coefficient, overlap coefficient after Manders (Manders, Verbeek and Aten, J. Microscopy 169:375-382, 1993) ∑ ∑ ∑ − Another parameter used to quantify colocalization in image pairs −...
Page 347 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Fig. 4-277 Image Display window, Profile display Fig. 4-278 Image Display window, Profile display 03/06 B 45-0021 e 4-285...
Page 348: 4.13.21.2 Function Elements
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.21.2 Function Elements The Profile toolbar contains the following function elements: Arrow (selection) button: Activates the mouse button for selection, resizing or movement of the profile line in the Image Display window.
Page 349 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Zoom button: Zoom function for profile diagram. Click and drag a rectangle over the area to be enlarged in the profile diagram; the selected area is enlarged on release of the mouse button.
Page 350: Display - 2.5 D
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.22 Display - 2.5 D This function allows to − display the two-dimensional intensity distribution of an image in an pseudo 3D mode −...
Page 351 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Fig. 4-279 Image Display window, 2.5 D display The Pseudo 3D toolbar contains the following function elements: Profile button Profile display (vertical polygon display). Setting of the Profile Distance between 1 and 20 using the slider.
Page 352: Display - 3D (Image Visart)
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.23 Display - 3D (Image VisArt) This optional function allows to − reconstruct and display 3 D fluorescence image stacks or time series of frames and stacks from the Image Display window −...
Page 353: 4.13.23.1 Shadow Projection
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Transparency projection Transparency rendering (voxel based) Basic button Advanced button Transparency rendering (voxel based) with textures Surface projection Basic button Surface rendering (voxel based)
Page 354 LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan The zoom wheel to the left of the Image Display window allows continuous zooming of the 3D reconstructed image. A click on the Any View button displays the 3D reconstruction image in a shadow projection where the viewing point can be defined.
Page 355 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss The following additional buttons are available in the Any View shadow projection mode: • After activation of the Frame button (below the image), a bounding box is drawn around the 3D reconstructed image.
Page 356 LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.23.2 Transparency Projection The transparency projection generates a transparent 3D reconstructed image. The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and animation function) are identical to those of the Any View function of the shadow projection and are operated in the same way.
Page 357: 4.13.23.3 Surface Rendering
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.23.3 Surface Rendering The surface rendering generates surface rendered 3D reconstructed images. The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and animation function) are identical to those of the transparency projection and are operated in the same way.
Page 358: Appearance (Settings)
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.23.4 Appearance (Settings) The Appearance button opens the 3D Rendering window. This window allows settings for Light, Material, Background and Projection properties to be defined for all 3D projection modes.
Page 359: 4.13.23.5 Series
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.23.5 Series The Series button opens the Render Series window. This window allows settings for the axis to be used for rotation of the 3D reconstructed images.
Page 360 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Start and End mode In the Start and End mode, the image is reconstructed between a start position and an end position.
Page 361: Display - Topography For Lsm
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.24 Display - Topography for LSM This optional function allows to − process, display and measure topographic information. − use frame Z Stacks − and frame Z Stacks over time The Topo function is mainly used for applications in material research and industry.
Page 362: 4.13.24.1 Channel Selection
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan The Topography toolbar contains the following function elements: The selection of a channel to be used. Channels buttons Generate buttons The selection of the mode of calculation of the topography image...
Page 363: 4.13.24.3 Topography Thresholds
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss First / Last • Click on the First button to calculate the topography surface by using the first slice coming from the top, where the intensity reaches the value defined by the lower intensity threshold.
Page 364: 4.13.24.4 Processing By Filtering
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Height threshold Click on the Height button to calculate the topography surface by using the lower and the upper height thresholds for image display. Use of...
Page 365 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss • To investigate the effects of various filter modes, select one of the 3D display modes (Profiles, Grid, Filled or Shaded) from the Display button bar.
Page 366 LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Changing the topography geometry The three buttons in the Geometry button bar allow the surface geometry to be changed. Inverse surface. Depths change to heights, and vice versa.
Page 367: 4.13.24.5 Display Modes
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.24.5 Display Modes The three buttons in the Display button bar allow stacks to be displayed in the 2D, Iso-Lines or 3D display mode.
Page 368 LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan The additional function elements of the Iso-Lines display mode have the following meaning: Line Dist. slider: Changes the distance of the iso-lines. Line Offset slider: Setting of the height level where the Iso-Lines display starts.
Page 369 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss The image viewing angle is set as follows: Setting directly in the image • Click in the image and hold down the mouse button. The perspective is changed by moving the mouse button in horizontal or vertical direction.
Page 370: Context Menu Of The 3D Display Mode
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.24.6 Context Menu of the 3D Display Mode • Click in the Image Display box with the right mouse button to open the context menu.
Page 371 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Export x,y,z- triples item This option is available in the Profiles and Grid 3D display modes. Use the function to save the Profiles or Grid data as a text file.
Page 372 LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Show processing parameters After selection of the Show processing parameters function, a reporting of the following applied topo processing functionality is displayed on the right-hand side of the Image Display window: −...
Page 373 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Save Calculation Parameters Topo routines can be saved and reloaded as tgp-files. These files include settings for: − reconstruction mode − intensity threshold − filters (including FFT) −...
Page 374 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Defining a specific Shading Model • Open the 3D Rendering window. • Click on the Define button. • Change the parameters of the topography using the appropriate sliders.
Page 375 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Projection panel Determines the reflective properties of a sample. View angle Determines the perspective, 0.0 parallel projection, central projection Zoom function, zoom in, zoom out Distance Fig.
Page 376 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan (11) Renderer item This option is only available in the Shaded 3D display mode. After selection of the Renderer item, the 3D Renderer window appears.
Page 377: 4.13.24.7 Measurement Functions
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.24.7 Measurement Functions The topography measurement functions are activated via the Measure button bar. The measurement functions can be performed in the 2D or 3D display mode.
Page 378 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Fig. 4-303 Topography display: 2D - Profile Additional Table / Profile buttons The additional Table / Profile buttons have the following functions: Show button: The profile is displayed in the form of a table at the bottom below of the Image Display window.
Page 379 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Activating the Profile measurement mode If you click on the button, the Profile window with the tools of the profile measurement mode appears. This window can be moved as required over the entire screen.
Page 380 Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Open Polyline button: Creation of an open polyline figure in the profile diagram. Display of the length of the line figure. First click sets the starting point, any further click adds another line, click with the right mouse button ends the procedure.
Page 381 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss z Histo measurement mode in 2D display • Click on the Diagram button in the Measure button bar. Click on the z Histo button in the additional button bar now displayed.
Page 382 LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Grad. Histo measurement mode in 2D display • Click on the Diagram button in the Measure button bar. Click on the Grad. Histo button in the additional button bar now displayed.
Page 383 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Calculation of roughness parameters The following roughness parameters are calculated (e.g. for a Y-section) − Mean height of all profile height values R ∑...
Page 384 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Classification of topography in 5 equal area elements (rectangles in the 2D mode) − average roughness depth R − − − −...
Page 385 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Calculation of roughness parameters The following roughness parameters are calculated: − Mean height of all surface height values S ∑ ∑ − ⋅ ⋅...
Page 386 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Classification of topography in 25 equal area elements (rectangles in the 2D mode) − average roughness depth S − − ⋅ ⋅ ⋅...
Page 387: 4.13.24.8 3D Measurement Functions
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.24.8 3D Measurement Functions Volume measurement mode (Flood function) • Use the 3D button to select the required 3D display of the stack. • Click on the Volume button in the Measure button bar.
Page 388 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Parameters The following parameters are calculated: height level (selectable with the Z-Threshold and Fill Level sliders). The setting of this value influences the following parameters.
Page 389 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Profile measurement mode in 3D display This function is performed in the same way as in the 2D display mode, with the following exceptions:...
Page 390 LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Curve of tp measurement mode in 3D display This function is performed in the same way as in the 2D display mode. Before determination of the tp bearing portion, individual peaks (noise, steep slopes) must be eliminated.
Page 391: 4.13.24.9 Export Data
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.24.9 Export Data − multiple profiles (Rel. 3.2) − single profile − parameters − topography as matrix − topography as triples 4.13.24.10 Topo ReUse Topo routines can be saved and reloaded as tgp-files (TopoGraphic Parameters).
Page 392: Display - Prev
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.25 Display - Prev. This function allows to − compose images, graphs and text for printing − use any image format − change fonts and line width in graphs via context sensitive menus The settings of Chan, Zoom, Slice, Contr and Palette apply.
Page 393: 4.13.25.1 Context Menu For Scan Information Text
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.25.1 Context Menu for Scan Information Text The context menu (right mouse button) allows to vary the output of the scan info . • Click with the right mouse button. A context menu with the options Color and Font is displayed.
Page 394: Display - Info
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.26 Display - Info This function allows to − display the parameters used during image acquisition of the image(s) displayed in the Image Display window −...
Page 395: Additional Display Mode In Time Series
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.27 Additional Display Mode in Time Series 4.13.27.1 Display - Mean This function allows to − display the intensity time diagram (mean intensity in user defined ROIs over time) −...
Page 396 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Fig. 4-313 Image Display window, Mean ROI display for time series of Z Stack The Mean of ROIs toolbar contains the following function elements: ROIs selection box: Display of the ROIs used during scanning of the time series and of the other ROIs available in the system.
Page 397 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Bezier and 3D Bezier button: Activates the Bezier figure drawing mode. The first click sets the starting point, each additional click adds a further line, a double-click on the starting point closes the figure and ends the procedure.
Page 398 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Buttons for diagram display: − 1 button: Intensity values for ROIs and channels are shown in one diagram. − Chan button: Intensity values are shown separately for each channel.
Page 399: Additional Display Modes In Lambda Mode
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.28 Additional Display Modes in Lambda Mode The following display modes of a Lambda Stack are available: − Display - Coded − Display - Max −...
Page 400: 4.13.28.3 Display - Mean
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan 4.13.28.3 Display - Mean This function allows to − display the intensity Lambda diagram (mean intensity in user defined ROIs over Lambda) −...
Page 401 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Rectangle button: Activates the rectangle drawing mode. Click and hold down the mouse button, drag the rectangle in any direction, release the mouse button to end the procedure.
Page 402 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Buttons for Table functions : Copy Table button: The table of intensity values over Lambda is copied to the clipboard. Show Table button: The table of intensity values over Lambda is displayed on the bottom left of the Image Display window.
Page 403: Functionality For Emission Fingerprinting
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss Performs the unmixing with the selected spectra. Linear Unmixing button Apply to hardware button Sets the parameters for the META detector used in binning mode as specified in the settings ChS1, ChS2, ChS3 ...
Page 404 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan • Deselect of individual channels in Lambda Stack affects: − Display in Lambda Coded and Lambda max modes, if Chan button in Select toolbar activated −...
Page 405: 4.13.28.5 Automatic Component Extraction
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Display and Analysis of Images Carl Zeiss 4.13.28.5 Automatic Component Extraction The use of this function permits the automatic search for the individual reference spectra in a lambda stack. Open / Close the Mean of ROI window •...
Page 406 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Display and Analysis of Images LSM 510 META DuoScan Little to moderate spatial overlap of emission signals signals is required Restriction: to obtain good results. Solution for Emission Fingerprinting in cases where reference spectra are Benefit: not accessible via single-labeled controls.
Page 407: Image Optimization
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Image Optimization Carl Zeiss 4.14 Image Optimization 4.14.1 Single Channel Described below is the example of the acquisition of an image, using an excitation wavelength of 543 nm and a fluorescence emission range above 560 nm. The HFT 488/543 is used as the main dichroic beam splitter.
Page 408 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Image Optimization LSM 510 META DuoScan • Click on the icon, activate the 543 nm laser line and click on Line Active . If required, deactivate other laser lines which are not needed.
Page 409 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Image Optimization Carl Zeiss • In the Main menu click on the Scan button in the Acquire subordinate toolbar. − This opens the Scan Control window. • Click on the Mode button.
Page 410 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Image Optimization LSM 510 META DuoScan • Click on the Channels button. − This displays the preset parameters of the configuration loaded. • Click on the Find button. Make sure to position the slider correctly.
Page 411: Pinhole / Detector Gain / Ampl. Offset / Ampl. Gain
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Image Optimization Carl Zeiss 4.14.1.2 Pinhole / Detector Gain / Ampl. Offset / Ampl. Gain • In the Scan Control window, click on the Cont. button (see Fig. 4-326). − This starts a continuous scan.
Page 412 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Image Optimization LSM 510 META DuoScan • Click on the Palette button in the Select image processing toolbar. − This opens the Color Palette window. • In the Color Palette List panel, click on the Range Indicator item.
Page 413 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Image Optimization Carl Zeiss If the image is not bright enough, it appears blue on the screen. • On the Channel Settings panel of the Scan Control window, set the PMT (photomultiplier) gain with the Detector Gain slider.
Page 414: Scan Speed, Scan Average And Pixel Depth
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Image Optimization LSM 510 META DuoScan 4.14.1.3 Scan Speed, Scan Average and Pixel Depth 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.
Page 415: Multiple-Channel
LSM 510 OPERATION IN EXPERT MODE LSM 510 META Image Optimization Carl Zeiss 4.14.2 Multiple-channel 4.14.2.1 Requirements • The suitable lasers are on. • The specimen has been positioned and focused for scanning. • Click on the LSM button in the Acquire subordinate toolbar of the Main menu.
Page 416: Image Optimization
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Image Optimization LSM 510 META DuoScan Fig. 4-335 Scan control and Image Display windows As a rule, the first scanned image (Pre-Scan) is not ideal, since the photomultiplier is not matched to the light output.
Page 417: Extension Of The Lsm 510 Or Lsm 510 Meta With Lsm Duoscan
Extension of the LSM 510 or LSM 510 META with LSM DuoScan The LSM 510 and LSM 510 META can be equipped with a second scanner, the LSM DuoScan . For optimal results the scan fields of the two scanners must be aligned. This procedure is described in chapter 4.8.4.1 The additional equipment results in different screen contents of the Bleach control menu (see chapter 4.5.7).
Page 418: 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. 4-337 LSM 510 Switchboard menu •...
Page 419: Shut Down The Windows Operating System
Set the REMOTE CONTROL switches “System/PC” and “Components” or the Main switch on the System Electronic Rack and the power supply switch of the Ar Laser to position " OFF " after 5 minutes. This puts your LSM 510 microscope system, including the computer, off power. 03/06 B 45-0021 e...
Page 420: Software And Hardware Options
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Software and Hardware Options LSM 510 META DuoScan 4.17 Software and Hardware Options This section describes optional software and hardware configurations. Depending on your configuration, the content of dialogue and function may differ.
Page 421 LSM 510 OPERATION IN EXPERT MODE LSM 510 META Software and Hardware Options Carl Zeiss If your configuration does not include the "Topography " software package, the following functions are not available: − Topo button in the Image Display window after acquisition of image stacks If your configuration does not include the "Macro Recorder and Editor"...
Page 422: Hardware
OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Software and Hardware Options LSM 510 META DuoScan 4.17.2 Hardware Depending on whether the following hardware components are available or not, the content of the screens may differ: − Piezo objective focusing device −...
Page 423: Courses On "How To Operate The System In An Optimized Way
4.18 Courses on "How to Operate the System in an Optimized Way" Carl Zeiss is offering training courses on how to operate the system in an optimized way. − Courses are held in our application center in Jena, Germany. − Courses are held in English or German language, respectively.
Page 424 OPERATION IN EXPERT MODE LSM 510 DuoScan Carl Zeiss Courses on "How to Operate the System in an Optimized Way"LSM 510 META DuoScan 4-362 B 45-0021 e 03/06...
Page 425 LSM 510 LSM 510 META Contents Carl Zeiss CHAPTER 5 VBA PROGRAMMING FOR LSM CONTENTS Page VBA PROGRAMMING FOR LSM ..................5-2 VBA Programming ......................5-2 5.1.1 General Syntax........................5-2 5.1.2 Working with Files ......................5-2 5.1.3 Access to Windows API Function and external DLLs ............5-4 Programming for LSM ......................5-8...
Page 426: Vba Programming For Lsm
VBA Programming LSM 510 DuoScan Carl Zeiss General Syntax LSM 510 META DuoScan VBA PROGRAMMING FOR LSM VBA Programming 5.1.1 General Syntax Object oriented programming Class Modules 5.1.2 Working with Files I/O operations, file operations are implemented in VBA Use of the implemented functions...
Page 427 LSM 510 VBA Programming LSM 510 META Working with Files Carl Zeiss Example: Type CALIBRATION_PARAM lambda As Long angle As Double Translation As Double End Type Function DataFromCalibFile(strGetFile As String, calibArray() As CALIBRATION_PARAM) As Integer Dim hFile As Long Dim strLine As String...
Page 428: Access To Windows Api Function And External Dlls
VBA Programming LSM 510 DuoScan Carl Zeiss Access to Windows API Function and external DLLs LSM 510 META DuoScan 5.1.3 Access to Windows API Function and external DLLs - with Type Libraries - with Declare Statements API Text Viewer searches WIN32API.TXT...
Page 429 LSM 510 VBA Programming LSM 510 META Access to Windows API Function and external DLLs Carl Zeiss Example: Declare Sub Sleep Lib "kernel32" (ByVal dwMilliseconds As Long) Declare Function RegOpenKeyEx _ Lib "advapi32.dll" Alias "RegOpenKeyExA" _ (ByVal hKey As Long, ByVal lpSubKey As String, _...
Page 430 VBA Programming LSM 510 DuoScan Carl Zeiss Access to Windows API Function and external DLLs LSM 510 META DuoScan Function FServerFromDescription(strName As String, strPath As String) As Boolean Dim lngResult As Long Dim strTmp As String Dim hKeyServer As Long...
Page 431 LSM 510 VBA Programming LSM 510 META Access to Windows API Function and external DLLs Carl Zeiss lngResult = RegCloseKey(hKeyServer) hKeyServer = 0 i = Len(strPath) Do Until (i = 0) If (VBA.Mid(strPath, i, 1) = "\") Then strPath = Left(strPath, i - 1)
Page 432: Programming For Lsm
Programming for LSM LSM 510 DuoScan Carl Zeiss Object structure LSM 510 META DuoScan Programming for LSM 5.2.1 Object structure Application Lsm5Application Lsm5 Lsm5VbaProject CpAmplifiers Lsm5Info CpCollimators Lsm5Tools CpFilterSets Lsm5Constants CpFocus CpIntegrators Lsm5Options CpLamps Lsm5Hardware CpLaserLines CpLasers DsGuidedModeDatabase CpObjectiveRevolvers DsFluorescenseDatabase...
Page 433 LSM 510 Programming for LSM LSM 510 META Object structure Carl Zeiss To search the object structure use the object browser. 03/06 B 45-0021 e...
Page 434: Differences In Access To Lsm Hardware With Lsm5Hardware Object / Dsrecording Object
Programming for LSM LSM 510 DuoScan Carl Zeiss Differences in access to LSM Hardware with … LSM 510 META DuoScan 5.2.2 Differences in access to LSM Hardware with Lsm5Hardware Object / DsRecording Object Object Browser Project / Library LsmVba auswählen und Objekt Struktur untersuchen...
Page 435 LSM 510 Programming for LSM LSM 510 META Differences in access to LSM Hardware with … Carl Zeiss For lI = 0 To TD.DetectionChannelCount - 1 Set DetS = TS.DetectionChannelObjectByIndex(lI, success) Set DetD = TD.DetectionChannelObjectByIndex(lI, success) DetD.Filter1 = DetS.Filter1 DetD.Filter2 = DetS.Filter2 DetD.DetectorGain = DetS.DetectorGain...
Page 436 Programming for LSM LSM 510 DuoScan Carl Zeiss Differences in access to LSM Hardware with … LSM 510 META DuoScan - Access with Lsm5Hardware Object Status will be updated at Scan Start to current recording parameters Private Sub FillPinholeList() Dim count As Long...
Page 437: Access To Hidden Interface
LSM 510 Programming for LSM LSM 510 META Access to hidden Interface Carl Zeiss 5.2.3 Access to hidden Interface - When do I need to use the hidden Interface? - How do I find the desired Object / Method? Open Tool / References Browse CP.dll search, open...
Page 438: Access To Scanned Pictures
Programming for LSM LSM 510 DuoScan Carl Zeiss Access to scanned pictures LSM 510 META DuoScan 5.2.4 Access to scanned pictures Example: Function ChannelIndexFromChannelName(ChannelName As String, ChannelIndex As Long) Dim num As Long, num1 As Long, num2 As Long Dim i As Long...
Page 439 LSM 510 Programming for LSM LSM 510 META Access to scanned pictures Carl Zeiss Function GetAveragePixel(ScanDoc As DsRecordingDoc, ChannelName As String, mean As Double) Dim line As Variant Dim x As Long, xmax As Long Dim y As Long, ymax As Long...
Page 440: Backup Recording
Programming for LSM LSM 510 DuoScan Carl Zeiss Backup Recording LSM 510 META DuoScan 5.2.5 Backup Recording Dim Recording As DsRecording Dim BackupRecording As DsRecording Dim ScanDoc As DsRecordingDoc Set Recording = Lsm5.DsRecording 'create a backup recording Set BackupRecording = Lsm5.CreateBackupRecording Success = BackupRecording.Copy(Recording)
Page 441: Events
LSM 510 Programming for LSM LSM 510 META Events Carl Zeiss 5.2.6 Events - What are Events good for? - Mouse Events - Scan state events - Hardware Events Private Sub Lsm5VbaDoc_SystemEvent(ByVal EventNr As Long, ByVal Param As Variant) Dim x As Long...
Page 442 Programming for LSM LSM 510 DuoScan Carl Zeiss Events LSM 510 META DuoScan 5-18 B 45-0021 e 03/06...
Page 443 LSM 510 ROUTINEMODE AND TOOLS LSM 510 META Contents Carl Zeiss CHAPTER 6 ROUTINE MODE AND TOOLS CONTENTS Page ROUTINE MODE AND TOOLS..................6-2 Routine Mode ........................6-2 6.1.1 General..........................6-2 6.1.2 Starting the Routine Mode ....................6-2 6.1.3 Application of Examination Methods in the Routine Mode ..........6-4 6.1.3.1...
Page 444: Routine Mode And Tools
6.1.1 General The Routine Mode of the LSM 510 software permits the fast and easy acquisition of scanning images by using time-tested 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.
Page 445 LSM 510 Routine Mode LSM 510 META Starting the Routine Mode Carl Zeiss In addition to the three standard examination methods contained in the LSM 510 software package, required number further examination methods can be exported from the Expert Mode to the Routine Mode.
Page 446: Application Of Examination Methods In The Routine Mode
LSM 510 DuoScan Carl Zeiss Application of Examination Methods in … LSM 510 META DuoScan The method taken over from the Expert Mode appears, with the entered name, in the Methods panel of the Select Method To Add To List window, though without an icon.
Page 447: Acquisition Of A Frame
LSM 510 Routine Mode LSM 510 META Application of Examination Methods in … Carl Zeiss 6.1.3.1 Acquisition of a Frame Selection of the examination method Standard Examination Methods • Click on the button of the required standard examination method, e.g. FITC / Rhodamine.
Page 448 LSM 510 DuoScan Carl Zeiss Application of Examination Methods in … LSM 510 META DuoScan • When you have completed your microscope settings, click on the Close button to close the Stage and Focus Control window. • Click on the Next button.
Page 449 LSM 510 Routine Mode LSM 510 META Application of Examination Methods in … Carl Zeiss Noise Reduction panel In the Noise Reduction panel, the scan speed and, depending on it, the scan time can be influ- enced. The longer the scan time, the better the noise Fig.
Page 450 Carl Zeiss Application of Examination Methods in … LSM 510 META DuoScan Function buttons in the Routine Mode - Image Setup window The selected scan mode is displayed at the top right of the Image Setup window for checking purposes.
Page 451: Acquisition Of A Z Stack
Expert Mode. • 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. 6-12 Image Save, Print & Export window 6.1.3.2...
Page 452 LSM 510 DuoScan Carl Zeiss Application of Examination Methods in … LSM 510 META DuoScan Routine Mode - Image Stack Setup window The buttons arranged on the right in the Image Stack Setup window have the following functions: New Image button Opens a new image window.
Page 453 LSM 510 Routine Mode LSM 510 META Application of Examination Methods in … Carl Zeiss Optical Slice: The Optical Slice window 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 454 Carl Zeiss Application of Examination Methods in … LSM 510 META DuoScan In case a line scan in Z is performed the following function buttons are available in the Line toolbar of the image created using the Line Select button Line Sel:...
Page 455 LSM 510 Routine Mode LSM 510 META Application of Examination Methods in … Carl Zeiss • Then click on the Range button. − The XZ-scan will be performed and displayed in the image window. At the same time, the position of the current slice is shown with a green line and the positions of the first and last slice with two red lines.
Page 456: Acquisition Of A Time Series
• Click on Save or Save As to store the acquired stack image. • Click on Finish to exit the Routine Mode and return to the LSM 510 Switchboard menu. 6.1.3.3 Acquisition of a Time Series A time series is recorded in the Routine mode in the same way as a frame.
Page 457 LSM 510 Routine Mode LSM 510 META Application of Examination Methods in … Carl Zeiss The following functions are available on the right side of the this window: New Image button Opens a new image window. Start T button Starts the time series.
Page 458 LSM 510 DuoScan Carl Zeiss Application of Examination Methods in … LSM 510 META DuoScan Starting a time series via the time • To start a time series via the time, the Start button must be pressed first. − Waiting for Start Time will be displayed in the status line. The time series is started when the starting time has been reached.
Page 459 LSM 510 Routine Mode LSM 510 META Application of Examination Methods in … Carl Zeiss • The interval to be used during the time series is set to a default value by activating a Time button. The time displayed on the time buttons can be changed as follows, if required: •...
Page 460 The also available Save and Save As buttons permit scanned images to be stored in a database (see chapter 4). • Click on Finish to exit the Routine Mode and return to the LSM 510 Switchboard menu. Fig. 6-24 Image Save, Print & Export window 6-18...
Page 461: Tools
The Change Filters tool is used to update the filter data in the software after a change of filters in the reflector turret. • Close the LSM 510 software program. • Insert the new filter module in the reflector turret.
Page 462 The Edit Filter List function permits updating of the filter data in the software after a change of filters on the stand. • Close the LSM 510 software program. • Double-click on the Change Filters icon on the desktop. • Click on the Edit Filter List button in the Emission Filter &...
Page 463 • Click on the Store button to accept the new settings. • Click on the Close button to close the Emission Filter & Beam Splitter Control window. When you start the LSM 510 software, the filter data are updated. 03/06...
Page 464: Stand Select
If this is not possible, proceed as follows: • Close the LSM 510 software program and double-click on the Stand Select icon on the desktop. − The Select Stand Database window appears on the screen. The currently used database is displayed in the Database box.
Page 465: Lsm Image Browser
When images are opened, image processing functions of the LSM 510 software are available to a limited extent (Chan, Zoom, Contr, Palette, Copy, Save, Save As, xy, Split xy, Prev, Info). • Click on the LSM Image Browser icon on the desktop of the PC. The Zeiss LSM Image Browser main menu is opened.
Page 466: Lsm Image Examiner
LSM Image Browser, plus the 3D functions and selected Process functions of the Expert Mode of the LSM 510. When images are opened, a large scope of the image processing functions of the LSM 510 software is available (for further details see in chapter 4).
Page 467 LSM 510 3D FOR LSM LSM 510 META Contents Carl Zeiss CHAPTER 7 3D FOR LSM CONTENTS Page 3D for LSM ........................7-2 Overview and Explanations....................7-2 7.1.1 The Image Sequence ......................7-2 7.1.2 The Image Properties......................7-3 7.1.3 Memory Usage........................7-3 User Interface........................7-3 7.2.1 Introduction ........................7-3...
Page 468: For Lsm
Voxel 7.1.1 The Image Sequence 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 469: The Image Properties
LSM 510 3D FOR LSM LSM 510 META User Interface Carl Zeiss 7.1.2 The Image Properties Every image sequence has its own set of properties. They contain the scaling and the scaling units. The scaling and its units are required for 3D reconstruction and measurement. If a sequence of LSM-TIFF images is read in, the image properties are loaded automatically from the file header and allocated to the image properties of the new image sequence.
Page 470: Main Window
3D FOR LSM LSM 510 DuoScan Carl Zeiss User Interface LSM 510 META DuoScan Display window This window is used to display image sequences. Display window Fig. 7-3 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.
Page 471 LSM 510 3D FOR LSM LSM 510 META User Interface Carl Zeiss 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 472 3D FOR LSM LSM 510 DuoScan Carl Zeiss User Interface LSM 510 META DuoScan Scrap Selects or deletes objects of a defined size. Fill Holes Fills holes in objects. View Menu Set Channel Colour The colour and the weight of the single channels can be defined.
Page 473 LSM 510 3D FOR LSM LSM 510 META User Interface Carl Zeiss 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 474 3D FOR LSM LSM 510 DuoScan Carl Zeiss User Interface LSM 510 META DuoScan Player This function plays back the sequential images of an image sequence. Fig. 7-4 The image sequence is displayed in the Display window. The display process is working as a background task;...
Page 475 LSM 510 3D FOR LSM LSM 510 META User Interface Carl Zeiss 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 476 3D FOR LSM LSM 510 DuoScan Carl Zeiss User Interface LSM 510 META DuoScan Set Channel Colour This function sets the colour and weight for the channels. Fig. 7-5 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 477: Functions
Fig. 7-6 The individual files of a Zeiss TIFF image sequence are read and saved as an image sequence in image memory. In addition, the image properties are read out of the TIFF files and allocated to the image sequence Input.
Page 478 LSM 510 META DuoScan 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.
Page 479 By choosing the Carl Zeiss Vision file format "KE Images (*0.img)", two files per channel are saved. On one hand the Carl Zeiss Vision type image sequence file, on the other hand the file with the image properties. One pair of files is written per channel. They are numbered automatically, starting with zero.
Page 480: Functions In The Edit Menu
3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan 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. From a multichannel sequence any channel status (on or off) combination can be defined.
Page 481 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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 482 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan On the Delete Channel tab sheet channels of the Input 1 image sequence can be selected to delete channels. Fig. 7-10 This operation might save time and memory for further processing if not all channels are needed.
Page 483: Functions In The Process Menu
LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 7.3.3 Functions in the Process Menu Arithmetics - Add This function adds two image sequences. Fig. 7-11 The Add tab sheet of the Arithmetics dialog window must be selected.
Page 484 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Arithmetics - Subtract This function subtracts two image sequences. Fig. 7-12 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 485 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Contrast - Interactive This function allows interactive changes of the contrast of an image sequence. Fig. 7-13 The Interactive tab sheet of the Contrast dialog window must be selected.
Page 486 Carl Zeiss Functions LSM 510 META DuoScan The Output histogram shows the resulting histogram. The horizontal axis represents the grey values from 0 to the maximum, which is either 255 or 4095, depending whether the input is 8 bit or 12 bit. The vertical axis represents the pixel count.
Page 487 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Contrast - Automatic This function scales the grey values of an image sequence to the maximum possible range. Fig. 7-14 The Automatic tab sheet of the Contrast dialog window must be selected.
Page 488 LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan The Output histogram shows the resulting histogram. They are not editable. The horizontal axis represents the grey values from 0 to the maximum, which is either 255 or 4095, depending whether the input is 8 bit or 12 bit.
Page 489 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Contrast – Linearize This function scales a range of grey values of an image sequence to equal area fractions in the histogram. Fig. 7-15 The Linearize tab sheet of the Contrast dialog window must be selected.
Page 490 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Parameters: Image Input image sequence Output Output image sequence SkipBlack 0 - Grey value black is ignored 1 - Grey value black is taken into account Input L...
Page 491 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Morphology The following four functions perform basic operations of mathematical morphology on image sequences. Fig. 7-17 As generalization of the morphology of two-dimensional images to three dimensions the structural elements are small volumina.
Page 492 Carl Zeiss Functions LSM 510 META DuoScan 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 493 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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 494 Carl Zeiss Functions LSM 510 META DuoScan 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 495 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Morphology - Erode This function erodes structures in an image sequence. Fig. 7-20 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 496 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Morphology - Dilate This function dilates structures in an image sequence. Fig. 7-21 In the Morphology dialog window, the tab sheet Dilate must be selected. Dilation makes bright regions larger on a dark background. It also results in the filling of gaps and smoothing of small contour details.
Page 497 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Morphology - Open This function carries out an opening. Fig. 7-22 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 498 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Morphology - Close This function carries out a closing. Fig. 7-23 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 499 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Segment - Interactive This function carries out a grey value segmentation by means of thresholding. Fig. 7-24 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 500 Carl Zeiss Functions LSM 510 META DuoScan 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 501 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Segment - Automatic The function carries out an automatic grey value segmentation by means of thresholding. Fig. 7-25 The Automatic tab sheet of the Segment dialog window must be selected. Segmentation is especially used to generate binary regions.
Page 502 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Parameters: Input Input image sequence Output Resulting image sequence Colour Green - Selected interval is displayed in green Blue/Red - Grey values below the selected interval are displayed in blue, grey...
Page 503 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Boolean - And This function carries out a bit-by-bit And calculation for the image sequences Input 1 and Input 2. Fig. 7-26 The And tab sheet of the Boolean dialog window must be selected.
Page 504 Carl Zeiss Functions LSM 510 META DuoScan If one or both input sequences are multichannel sequences, any number or combination can be selected. The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from left to right.
Page 505 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Boolean - Not This function carries out a bit-by-bit negation of an image. Fig. 7-29 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 506 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Boolean - Mask This function masks a grey value image sequence. Fig. 7-30 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 507 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Scrap This function deletes or selects objects in a specified size range. Fig. 7-31 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 508 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Fill Holes This function fills holes in all objects. Fig. 7-32 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 509: Functions In The View Menu
LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 7.3.4 Functions in the View Menu Render - Surface This function displays an image sequence according to the gradient shading method. Fig. 7-33 The Surface tab sheet of the Render dialog window must be selected.
Page 510 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan 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 511 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss Grey High High grey value threshold of the region to be displayed Aperture Measure of the extent of the highlights Reflection Weight of the defuse brightness components in comparison to the highlights...
Page 512 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Render - Alpha This function displays an image sequence according to the alpha rendering method. Fig. 7-34 The Alpha tab sheet of the Render dialog window must be selected.
Page 513 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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 514 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Parameters: Input Input image sequence Output Resulting image sequence Number of Views Number of reconstructions to be calculated Angle X Angle of rotation on the X-axis, start position...
Page 515 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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 516: Functions In The Measurement Menu
3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan 7.3.5 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 517 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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...
Page 518 LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan 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 519 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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 520 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan 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.
Page 521 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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...
Page 522 Carl Zeiss Functions LSM 510 META DuoScan 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 523 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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 524 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan Automatic Object Measurement - General This function carries out an automatic measurement and labeling. Measured Object Features Measured Volume Features Fig. 7-40 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 525 LSM 510 3D FOR LSM LSM 510 META Functions Carl Zeiss 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.
Page 526 3D FOR LSM LSM 510 DuoScan Carl Zeiss Functions LSM 510 META DuoScan 7-60 B 45-0021 e 03/06...
Page 527 Filter Change in the Detection Beam Path of Channels 1 and 2 ........8-13 Piezo Objective Focussing Device ..................8-14 Specifications of Trigger-Interface LSM 510..............8-15 Monitor Diode .........................8-18 NLO Non Linear Optics Laser for LSM 510 NLO and LSM 510 META NLO......8-25 8.8.1 Laser Control Window .....................8-26 8.8.2 Configuration Control Window..................8-27...
Page 528: Annex
ANNEX LSM 510 DuoScan Carl Zeiss Beam Path Configuration Guide for ... LSM 510 META DuoScan ANNEX Beam Path Configuration Guide for Fluorescence Microscopy with the LSM 5 8.1.1 Optical elements in the Configuration Control window All wavelength values given in Nanometer [nm].
Page 529: Miscellaneous
LSM 510 ANNEX LSM 510 META Beam Path Configuration Guide for Fluorescence Microscopy ... Carl Zeiss 8.1.1.4 Miscellaneous • Plates do transmit light 100%. They are used for a correct beam guidance and will be set automatically. • Mirrors do deflect 100% over the whole spectral range and can be used to guide the emission light to selected detectors.
Page 530 LSM 510 DuoScan Carl Zeiss Beam Path Configuration Guide for ... LSM 510 META DuoScan • Check the available emission filters (EM 1- 4) for transmission of fluorescent light from the specimen, in order to identify the channels for detection.
Page 531: Multi Track Configuration
LSM 510 ANNEX LSM 510 META Beam Path Configuration Guide for Fluorescence Microscopy ... Carl Zeiss 8.1.3 Multi Track Configuration Multitracking is the method of choice for multi fluorescence imaging. It has the advantage to avoid artifacts based on emission crosstalk that occurs when using simultaneous excitation and detection.
Page 532: Beam Path Configuration For Multi Photon Excitation
LSM 510 DuoScan Carl Zeiss Beam Path Configuration Guide for ... LSM 510 META DuoScan Line and Frame Mode of Multitracking • Settings can be used for Line or Frame wise Multitracking. • In Line mode the lines are scanned in turns for all tracks with the corresponding laser lines turned on exclusively.
Page 533: Recommendations For Excitation Laser Lines And Emission Filters Of Dyes
LSM 510 ANNEX LSM 510 META Recommendations for Excitation Laser Lines and … Carl Zeiss Recommendations for Excitation Laser Lines and emission filters of dyes Laser line/HFT Emission/EM DAPI 364 or 405 > 385/420, max. at 461 EBFP 364 or 405 >...
Page 534 ANNEX LSM 510 DuoScan Carl Zeiss Recommendations for Excitation Laser Lines … LSM 510 META DuoScan Here you can note your specific combinations: Dyes Laser/HFT Example: Dyes Laser/HFT FITC/Cy3 488/543 BP 505-530 LP 560 B 45-0021 e 03/06...
Page 535: Configurations Overview
LSM 510 ANNEX LSM 510 META Configurations Overview Carl Zeiss Configurations Overview Package Count META Laser lines Laser lines optional Configuration Channels Detector (additional) 3 Channels F/R No ArMI 458/477/488/514, Ti:Sa HeNe 543, HeNe 633 700 - 1050 nm 23 META...
Page 536 ANNEX LSM 510 DuoScan Carl Zeiss Configurations Overview LSM 510 META DuoScan Configuration Main beam Secondary beam Secondary beam Secondary beam splitter (HFT) splitter 1 (NFT 1) splitter 2 (NFT 2) splitter 3 (NFT 3) NT 80/20 empty mirror empty...
Page 537 LSM 510 ANNEX LSM 510 META Configurations Overview Carl Zeiss Configuration Main beam Secondary beam Secondary beam Secondary beam splitter (HFT) splitter 1 (NFT 1) splitter 2 (NFT 2) splitter 3 (NFT 3) 25 META NT 80/20 empty mirror empty...
Page 538 ANNEX LSM 510 DuoScan Carl Zeiss Configurations Overview LSM 510 META DuoScan Configuration Main beam Secondary beam Secondary beam Secondary beam splitter (HFT) splitter 1 (NFT 1) splitter 2 (NFT 2) splitter 3 (NFT 3) NT 80/20 empty mirror empty...
Page 539: Filter Change In The Detection Beam Path Of Channels 1 And 2
LSM 510 ANNEX LSM 510 META Filter change in the Detection Beam Path of Channels 1 and 2 Carl Zeiss 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 the motor- controlled change between different filters for narrow-band or broad-band detection depending on the wavelength.
Page 540: Piezo Objective Focussing Device
ANNEX LSM 510 DuoScan Carl Zeiss Piezo Objective Focussing Device LSM 510 META DuoScan Piezo Objective Focussing Device For Axio Imager.Z1, Axio Imager.M1 and Axiovert 200 Range: 250 µm Minimum step size: 50 nm Speed: Piezo objective focussing device Slices Step size [µm]...
Page 541: Specifications Of Trigger-Interface Lsm 510
Specifications of Trigger-Interface LSM 510 / LSM 510 META Application: With the LSM 510 / LSM 510 META 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 542 ANNEX LSM 510 DuoScan Carl Zeiss Specification of Trigger-Interface LSM 510 … LSM 510 META DuoScan 15-point high density D-Type plug: Plug „A“ Signal name description SyncOUT0 Synchronous output SyncOUT1 Synchronous output SyncOUT2 Synchronous output SyncOUT3 Synchronous output Signal ground...
Page 543 LSM 510 ANNEX LSM 510 META Specification of Trigger-Interface LSM 510 / LSM 510 META Carl Zeiss Type/Voltage Range: − TTL signal level 3.3 V, CMOS low power consumption − 5.0 V tolerant input/output for interfacing with 5 V logic...
Page 544: Monitor Diode
LSM 510 META DuoScan Monitor Diode The monitor diode 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 to check the laser input in terms of power and noise.
Page 545 LSM 510 ANNEX LSM 510 META Monitor Diode Carl Zeiss Application examples: Checking the laser power This function is not automated so far. To qualitatively measure the laser power, the diode can be used in such a way that the gray level...
Page 546 ANNEX LSM 510 DuoScan Carl Zeiss Monitor Diode LSM 510 META DuoScan To use the monitor diode for ratio application, proceed as follows: • Click on the Ratio button. • Activate the ratio channel R1 or R2 in the Ratio...
Page 547 LSM 510 ANNEX LSM 510 META Monitor Diode Carl Zeiss The following image is an example of the reduction of correlated noise. The low frequency noise has been generated artificially. Fig. 8-8 Image Display window 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 512 x 512 pixels (noise without object information).
Page 548 LSM 510 DuoScan Carl Zeiss Monitor Diode LSM 510 META DuoScan 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 549 LSM 510 ANNEX LSM 510 META Monitor Diode Carl Zeiss Amplifier Gain The diode signal is set to the right range (gray level between 50 and 200 - 8 bit image / 750 and 3500 - 12 bit image) with the help of gray filters and amplifier gain.
Page 550 ANNEX LSM 510 DuoScan Carl Zeiss Monitor Diode LSM 510 META DuoScan 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 formula. Four types of formulas are offered when the R1 button is pushed.
Page 551: Nlo Non Linear Optics Laser For Lsm 510 Nlo And Lsm 510 Meta Nlo
These lasers are not operated via the LSM software. Refer to the operators manual of the manufacturer for using these lasers in combination with the LSM 510. However it is needed to indicate the tuned wavelength in the software to ensure the appropriate setting of the AOM frequency (see below).
Page 552: 8.8.1 Laser Control Window
ANNEX LSM 510 DuoScan Carl Zeiss NLO Non Linear Optics Laser for LSM 510 NLO … LSM 510 META DuoScan 8.8.1 Laser Control Window The Laser Control window shows the NLO laser as additional laser of the system. Operating a Chameleon laser •...
Page 553: 8.8.2 Configuration Control Window
LSM 510 ANNEX LSM 510 META NLO Non Linear Optics Laser for LSM 510 NLO and … Carl Zeiss Operating Verdi Mira or Millenia Tsunami lasers • For other lasers: The laser itself is not switched on when clicking On, this has to be done directly at the laser, but the software is informed that the laser is switched on and the laser safety shutter in the AOM box can be opened.
Page 554: Pinhole And Collimator Settings
ANNEX LSM 510 DuoScan Carl Zeiss NLO Non Linear Optics Laser for LSM 510 NLO … LSM 510 META DuoScan 8.8.3 Pinhole and Collimator Settings • The pinhole can be fully opened for maximum detection efficiency due to the focal excitation capabilities of the NLO effect (see Scan Control window, Channel Settings).
Page 555 LSM 510 ANNEX LSM 510 META Non Descanned Detection Carl Zeiss • Click Non-Descanned Detection button to change to Non-Descanned Detection. • Configure the NDD channels analog to the Descanned Detection mode. The configuration of multitracks is also possible for NDD applications, though not...
Page 556: Axiocam High Resolution Digital Cameras
ANNEX LSM 510 DuoScan Carl Zeiss AxioCam High Resolution Digital Cameras LSM 510 META DuoScan 8.10 AxioCam High Resolution Digital Cameras 8.10.1 High Resolution Microscopy Camera AxioCam HRm Rev.2/3.3V Cat. No 426511-0000-000, incl. digital interface and cable High Range Monochrome Number of Pixels: 1388 (H) x 1040 (V) = 1.4 Mega pixel...
Page 557: Microscope Camera Port Adapters For The Axiocam
LSM 510 ANNEX LSM 510 META AxioCam High Resolution Digital Cameras Carl Zeiss Dynamic Range: Typical 2000 : 1 @ 9 e readout noise Integration Time: 1 ms to several minutes Cooling: One stage Peltier cooling Optical Interface: C-Mount Size: about 11 cm x 8 cm x 6.5 cm (2.3"...
Page 558: List Of Key Words
And ............7-37 Dichroic beam splitters.......4-60 Or............7-37 Dilation......7-28, 7-30, 7-31, 7-32 Xor ............7-38 Display of images........4-253 Carl Zeiss Vision image sequences .....7-12 Display window ........7-4, 7-7 Camera color adjustment ......4-249 Dyes ............8-7 Camera detection........4-74 Channel ........4-81, 4-255 add, remove ......... 7-5, 7-15 Edit menu............7-5...
Page 559 LSM 510 ANNEX LSM 510 META List of Key Words Carl Zeiss Save display as ........7-13 Emission filter ........4-59, 8-7 Save image as ........7-12 Erosion ....7-27, 7-28, 7-29, 7-31, 7-32 Scrap ........... 7-41 Excitation......... 4-63, 4-71 Segment-automatic......7-35 Laser lines ..........8-7...
Page 560 ANNEX LSM 510 DuoScan Carl Zeiss List of Key Words LSM 510 META DuoScan Line ..........4-75, 6-13 creation of a .........4-97 Objective ..........4-240 Line selection..........4-88 Object features Lowpass filter ..........4-143 Densitometric........7-55 Geometric..........7-54 LSM 510 Switchboard ......4-363 Opening ............7-31 Options ......... 4-18, 4-218 Macro ...........
Page 561 Track configuration....4-55, 4-57, 4-58 Settings ...........4-222 Tracks panel, List of ........4-65 Settings for excitation ........4-67 Transmission ........... 4-353 Sharpness filter ........4-144 Trigger-interface LSM 510......8-18 Shut-down procedure......4-363 Turning power off........4-364 Single channel .........4-352 Single track..........4-55 Unmix............. 4-152 Single tracking.........
Page 562 ANNEX LSM 510 DuoScan Carl Zeiss List of Key Words LSM 510 META DuoScan Value Enter new value ........7-20 Mask grey value........7-40 Scale a range of grey values ....7-23 Scale grey value ........7-21 Vertical scale of histogram ......7-20 View menu..........7-6 Visual macro editor........4-209...
Page 563 Multiphoton Laser Scanning Microscopy Using the Zeiss LSM 510 META NLO Urechis caupo Serotonin positive nerve cells Written by Mary Dickinson, PhD Biological Imaging Center California Institute of Technology February 2002...
Page 565 Using the LSM 510 NLO direct coupled system: Practical considerations for ....optimal imaging....................9-13 9.3.1 Coupling an ultrafast laser to the LSM 510 NLO ...............9-13 9.3.2 Alignment of the ultrafast laser into the LSM 510 NLO scan head ........9-14 9.3.2.1 Quick alignment protocol....................9-14 9.3.2.2 Advanced alignment protocol ..................9-16 9.3.2.3...
Page 566 MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Contents LSM 510 META NLO B 45-0021 e 03/06...
Page 567: Multiphoton Laser Scanning Microscopy
MULTIPHOTON LASER SCANNING MICROSCOPY LSM 510 META NLO Preface Carl Zeiss MULTIPHOTON LASER SCANNING MICROSCOPY Preface Safety Considerations when using ultrafast lasers coupled to a microscope. Users operating ultrafast lasers have to observe all of the precautions specified in the Operating Manual for the laser and caution should be exercised when using the laser.
Page 568: Introduction To Multiphoton Laser Scanning Microscopy
MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Introduction to Multiphoton Laser Scanning Microscopy LSM 510 META NLO Introduction to Multiphoton Laser Scanning Microscopy Multiphoton laser scanning microscopy (MPLSM) has become an important technique in vital and deep tissue fluorescence imaging. In MPLSM, fluorescent molecules are excited by the simultaneous absorption of two or more near infrared (NIR) photons.
Page 569 MULTIPHOTON LASER SCANNING MICROSCOPY I LSM 510 META NLO ntroduction to Multiphoton Laser Scanning Microscopy Carl Zeiss While this appears conceptually simple, two difficulties, at the level of the fluorochrome, confound our understanding of this process. First, it is difficult to predict whether a molecule will efficiently absorb the two lower energy photons simultaneously.
Page 570: Increased Signal-To-Noise, Enhanced Vitality, And Deep Optical Sectioning In Mplsm
One-photon vs. Multiphoton improve optical sectioning. This can be done on an excitation. Two images of an LSM 510 NLO by carefully adjusting the collimation objective are shown for each lens and the z-position of pinhole 1 (Refer to the example.
Page 571: Drawbacks Of Using Nir Light For Microscopy
MULTIPHOTON LASER SCANNING MICROSCOPY I LSM 510 META NLO ntroduction to Multiphoton Laser Scanning Microscopy Carl Zeiss The lack of out-of-focus excitation greatly reduces bleaching, and therefore, photodamage, throughout the sample. This reduces damage caused by repeated or slow scans; however, photobleaching at the focal plane is still present.
Page 572: Achieving Efficient Multiphoton Excitation Using Ultrafast Lasers
MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Introduction to Multiphoton Laser Scanning Microscopy LSM 510 META NLO 9.2.4 Achieving Efficient Multiphoton Excitation using Ultrafast lasers In order to achieve efficient two- or three-photon excitation, the photons must collide with the molecule simultaneously.
Page 573: Optimizing The Peak Intensity Without Frying The Samples
MULTIPHOTON LASER SCANNING MICROSCOPY I LSM 510 META NLO ntroduction to Multiphoton Laser Scanning Microscopy Carl Zeiss The exact dimensions of A will vary greatly depending on the properties of the objective. In general, higher magnification, higher NA objectives will reduce A, similar to the way such objectives increase resolution.
Page 574 MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Introduction to Multiphoton Laser Scanning Microscopy LSM 510 META NLO 6 nm bandwidth 10 nm bandwidth Fig. 9-3 Effect of GVD on short pulses with a broad bandwidth. The broader the bandwidth , the more the pulse is stretched.
Page 575: Using The Lsm 510 Nlo Direct Coupled System: Practical Considerations For
Coupling an ultrafast laser to a laser scanning microscope is a fairly simple matter, if several key considerations are understood. Typically, the routing kit for coupling the LSM 510 NLO to the laser will consist of the following components: an Acousto-Optic Modulator (AOM), used to control the intensity of the beam and for beam blanking (allowing for region of interest (ROI) and bi-directional scanning and for performing Auto-Z-Brightness correction);...
Page 576: Alignment Of The Ultrafast Laser Into The Lsm 510 Nlo Scan Head
Alignment of the ultrafast laser into the LSM 510 NLO scan head The alignment of the laser is done by a Zeiss service engineer. However it might be needed to slightly adjust the beam coupling from time to time, depending on the room conditions. The protocols mentioned should only be performed if the user is confident with the technique, otherwise the coupling of the laser can be lost and a Zeiss service engineer is needed for realignment.
Page 577 MULTIPHOTON LASER SCANNING MICROSCOPY LSM 510 META NLO Using the LSM 510 NLO direct coupled system Carl Zeiss Fig. 9-5 (A) Setup for checking the beam alignment; (B) Optimal shape of the NIR laser spot Ideally this spot should be 5-7 mm in diameter and should be completely round and uniform. If the beam appears to be clipped (flat on one side), small adjustments in the routing mirrors can improve the alignment (see above).
Page 578: Advanced Alignment Protocol
MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Using the LSM 510 NLO direct coupled system LSM 510 META NLO 9.3.2.2 Advanced alignment protocol When the system is installed, the service engineer will establish alignment of the laser beam into the scan head by verifying the overlay between the NIR laser and one of the VIS lasers.
Page 579: Tips On Maintaining Alignment With The Scan Head
MULTIPHOTON LASER SCANNING MICROSCOPY LSM 510 META NLO Using the LSM 510 NLO direct coupled system Carl Zeiss Begin scanning using the FAST XY mode. An image of the grid should appear on the screen. If not, adjust the gain for each channel so that the output from both channels is visible. The two colors represent the reflection of the 543 nm line (green) and the reflection of the NIR laser (red) on the metallic grid surface.
Page 580 LSM 510 NLO and the microscope stand for a wavelength of 800 nm. The PTD effect is negligible if the pulse length is not much shorter than 100 fsec. It can be seen that the special IR corrected objectives show a smaller PTD in comparison to the standard UV/VIS corrected objectives.
Page 581 800 nm. (*) Propagation time difference (PTD) is calculated for the whole optical setup including the LSM 510 NLO and the microscope stand. Negative values indicate that pulses at the edge of the pupil are delayed with respect to pulses traveling on axis.
Page 582: Choosing Fluorescent Probes For Mplsm
MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Using the LSM 510 NLO direct coupled system LSM 510 META NLO While the Achroplan objectives clearly provide the best transmission curves with the longest working distance, other objectives have proven to be useful for multiphoton imaging. For instance, the Plan- Apochromat objectives have favorable properties for multiphoton imaging as Table 1 indicates.
Page 583 MULTIPHOTON LASER SCANNING MICROSCOPY LSM 510 META NLO Using the LSM 510 NLO direct coupled system Carl Zeiss The web-site shows a collection of excitation cross Blue/Cyan dyes sections for various dyes. Currently available dyes Alexa 350 (780 nm-800 nm) have multiphoton cross-section values ranging from 40-200 GM (see Xu, 2000).
Page 584: Samples Your Mother Should Have Warned You About
MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Using the LSM 510 NLO direct coupled system LSM 510 META NLO As mentioned above, photostability is an important factor when choosing a dye. Photobleaching is minimal in out-of-focus regions using MPLSM, but bleaching still occurs at the point of focus. In fact, some dyes that are relatively stable using one-photon excitation, have more rapid bleaching rates using MPLSM (Patterson and Piston, 2000).
Page 585 MULTIPHOTON LASER SCANNING MICROSCOPY LSM 510 META NLO Using the LSM 510 NLO direct coupled system Carl Zeiss Other molecules, such as salt crystals, can also focus light within tissues and intensify heating effects. This can also lead to imaging artifacts, perhaps due to signal produced via second harmonic generation (SHG).
Page 586 MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Using the LSM 510 NLO direct coupled system LSM 510 META NLO Artifacts can also occur in fixed tissue. Local heating effects can cause bubbles to form in the mounting media induce localized photochemistry in some mounting medias. It is important to test all mounting medias before use for multiphoton imaging.
Page 587: Troubleshooting Checklist
MULTIPHOTON LASER SCANNING MICROSCOPY LSM 510 META NLO Troubleshooting Checklist Carl Zeiss Troubleshooting Checklist Several key things can go wrong when imaging that will result in poor or no image being produced. Below is a checklist to help the user produce the best images possible.
Page 588: Poor Image Quality
MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss Troubleshooting Checklist LSM 510 META NLO 9.4.2 Poor image quality Lines or spaces across the image: This can result from an unstable mode-lock. CW breakthrough appears as blank lines in the image. Q-switching will result in wavy lines in the image. A service engineer from Choherent or Spectra need to check the laser.
Page 589: References
MULTIPHOTON LASER SCANNING MICROSCOPY LSM 510 META NLO References Carl Zeiss References Albota, M., Beljonne, D., Bredas, J.L., Ehrlich, J.E., Fu, J.Y., Heikal, A.A., Hess, S.E., Kogej, T., Levin, M.D., Marder, S.R., McCord-Maughon, D., Perry, J.W., Rockel, H., Rumi, M., Subramaniam, C., Webb, W.W., Wu, X.L., Xu, C.
Page 590 MULTIPHOTON LASER SCANNING MICROSCOPY Carl Zeiss References LSM 510 META NLO Xu, C. (2000) Two-photon Cross Sections of Indicators. In, Imaging Neurons: A Laboratory Manual. R. Yuste, F. Lanni, and A Konnerth, eds. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Page 591 LSM 510 and LSM 510 META Laser Scanning Microscopes Brief Operating Manual Release 4.0 March 2006...
Page 592 Introduction For your safety! Observe the following instructions: The LSM 510 and LSM 510 META laser scanning microscope, including its original − accessories and compatible accessories from other manufacturers, may only be used for the purposes and microscopy techniques described in this manual (intended use).
Page 593: Starting The System
Starting the System Switching on the LSM system • When set to ON the REMOTE CONTROL switch labeled System/PC provides power to the microscope and the computer. This allows to use the microscope and the computer without running the LSM Software (Fig. 1). •...
Page 594 Starting the LSM 5 software program • Double click the LSM 510 icon on the desktop of WINDOWS to start the LSM 5 software program. LSM 510 The LSM 510 Switchboard window appears on the screen (Fig. 3). Fig. 3 LSM 510 Switchboard menu •...
Page 595 Creating a database for acquired images • Click on the File button in the Main menu toolbar. The File subordinate toolbar appears in the Main menu. Fig. 5 Main menu with File subordinate toolbar • Click on the New button in the File subordinate toolbar. The Create New Database window appears.
Page 596: Setting The Microscope
• Click on the LSM button to set the microscope screen observation via laser excitation using the LSM 510 and software evaluation. Setting the microscope and storing the settings • Click on the VIS button for direct observation.
Page 597 Selecting an objective • Open the graphical pop-up menu by clicking on the Objective button (Fig. 10). • Click on the objective you want to select. The selected objective will automatically move into the beam path. Focussing the microscope for transmitted light •...
Page 598: Configuring The Beam Path And Lasers
Configuring the beam path and lasers • Click on the LSM button in the Acquire subordinate toolbar for laser scanning. Choosing the configuration Single Track − Use for single, double and triple labeling; simultaneous scanning only Advantage: faster image acquisition −...
Page 599 • Select the appropriate filters and activate the channels. • Click the Excitation button to select the laser lines attenuation values (transmission in %) in the displayed window. For the configuration of the beam path, please refer to the application-specific configurations depending on the used dyes and markers and the existing instrument configuration.
Page 600 • Click on the Multi Track button in the Configuration Control window (Fig. 15). • Click on the Descanned button if necessary. The following functions are available in the List of Tracks panel (Fig. 15). Modes Switch tracks after each Line button Tracks are switched during scanning line by line.
Page 601: Scanning An Image
Scanning an image • Click on the Scan button in the Acquire subordinate toolbar to open the Scan Control window. The Scan Control window appears (Fig. 16). Setting the parameters for scanning • Select Mode in the Scan Control window. •...
Page 602 In the Sum method, the pixel values of all scans are only added up, without a mean value being calculated. • Select the Number for averaging. Continuous averaging is possible in the Frame mode. In this case a Finish button for ending continuous averaging is displayed instead of the Cont.
Page 603 Image optimization Choosing a lookup table • Select Palette in the Image window of the scanned image (Fig. 19). The Color Palette window appears. • In the Color Palette List panel, click on the Range Indicator item (Fig. 20). The scanned image appears in a false-color presentation (Fig.
Page 604 Scanning a Z stack • Select Z Stack in the Scan Control window. • Select Frame if necessary. The Z Settings panel appears. • Select Mark First/Last on the Z Settings panel. • Click on the XY cont button. A continuous XY-scan of the set focus position will be performed.
Page 605: Storing An Image
Storing an image • Click on the Save or Save As button in the Image window or in the File subordinate toolbar of the Main menu. The Save Image and Parameter As window appears. Fig. 23 Save Image and Parameter As window •...

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Zeiss LSM 510 Manual

Chapter 1 Notes on Device Safety

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