Zeiss LSM 880 User manual

LSM 880
LSM 880 NLO
Operating Manual
October 2014
ZEN 2 (black edition)

INTRODUCTION
ZEISS LSM 880
II 000000-2071-464 10/2014 V_01
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.
The specifications are subject to change; the manual is not covered by an update service.
© Unless expressly authorized, forwarding and duplication of this document, and the utilization and
communication of its contents are not permitted. Violations will entail an obligation to pay
compensation.
All rights reserved in the event of granting of patents or registration of a utility model.
Issued by Carl Zeiss Microscopy GmbH
Carl-Zeiss-Promenade 10
07745 Jena, Germany
[e-mail]
www.zeiss.com/microscopy

INTRODUCTION
LSM 880 ZEISS
10/2014 V_01 000000-2071-464 III
How to make best use of the LSM 880 and LSM 880 NLO operating instructions:
A few symbols in these operating instructions will help you to recognize the nature and purpose of
information immediately:
The WARNING symbol warns against hazards for the user that might arise when operating the
laser.
This WARNING symbol warns against hazards from dangerously high voltages.
The
CAUTION symbol warns against faults and hazards that might arise during operation and
which might cause damage to the unit.
The NOTE symbol will help you to optimally solve your work problem. It represents a practical
tip which will help you to find out
which settings and methods are capable of improving or
accelerating a procedure.
The HOT SURFACE symbol warns against hazards for the user that might arise when touching
the lamp housing during operation.
The MAINS PLUG symbol remembers service personal to pull the mains plug before opening the
device housing.
Depending on the request, these operating instructions will supply you with various possibilities:
•If you want to know where to find certain general areas of information, refer to the following outline
of sections to get a general overview.
•You will find a detailed table of contents at the start of every chapter. There you will see at a glance
what topics are covered in detail.
Always remember: The time you invest in getting acquainted with the product will pay
for itself many times over in your application task.

INTRODUCTION
ZEISS LSM 880
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Contents
1
System Operation
In this section you will find the most important steps and procedures of the
LSM menu structure. The step-by-step description how to get an image will
be shown by
typical application examples including the WINDOWS graphic user environment.
2
Macros and Visual Basic
This section contains a description of the use of additional functions, e.g. maintenance,
macros.
3
Additional Software Tools
This section contains a description of the use of additional tools for setting the microscope.
4
Annex
The annex contains the Application-
specific Configurations, special notes and information for
using the LSM microscope.
5
Multiphoton Laser Scanning Microscopy with LSM Family Systems
This section provides detailed back ground information on Multiphoton Microscopy including
trouble shooting procedures.
6
Certification
7
Laser Safety Warning Labels
8
Introduction to Laser Scanning Microscopy
The section introduces you to the performance features of your LSM system.

IMPORTANT NOTES
LSM 880 ZEISS
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IMPORTANT NOTES FOR CHAPTER 1
•Visual Macro Editor does not allow saving .czi file format.
•Auto Z correction function works with laser powers smaller than 0.5 % but does not display the
changes even if Enable Test is active.
•Do not use "Double click in image" function for shuttle and find as this does move stage when
keeping the mouse button pressed.
•Processed PALM data do not show up as image in the Shuttle and Find View tab, use unprocessed
data instead.
•Do not use "Double click in image" function for shuttle and find as this does move stage even without
calibration data.
•Creating a new block moves the stage in Z to a lower position. Duplicate an existing block instead to
not lose the focus.
•Do not reuse images which have been acquired with polarization filters use for acquisition. The filters
cannot be removed from the beam path afterwards and ZEN has to be restarted if that is needed.
•The Z coordinate in the info view refers to the center position for Position Experiments or the first
image of the stack for Non-Position Experiments.
•Using Fast Z Line mode in combination with a Multitrack does result in wrong results. Do not check
fast Z line or avoid multi-tracking.
•Multi-positions will not be concatenated; the positions dimension is lost.
•Tuning a multiphoton laser with inbuilt dispersion compensation to a different wavelength for
bleaching might result in the first image(s) following the bleach event not being final as the GDD can
still be moving. This can be set via database. Contact your ZEISS representative on details if that
function is of interest for you.
•Using arrow, line or scale bar as graphics tool in non-processed Airyscan data (Airyscan View) does
change table parameters in an unspecified way. Use these tools only for processed data.
•Concatenating SR mode and VP mode acquired datasets using modify series or the concatenate macro
produces faulty results.
•Spot select for spot imaging or bleaching should only be used within one image container. Applying it
to images in different containers may result in malposition of the spots.
•Using the split mode of the 3D view does not work in combination with measurements in 3D. Use
either or for display or measurement.
•Do not use ILEX mode other than when Airyscan detector is set active.
•When using LSM and SIM imaging in multitrack mode, use the maximal pixel resolution for the
camera to be able to apply a fit function to the SIM image.
•Particle tracking requires non processed PALM data as input. Processed data produce no results.
•For wide-field imaging, set the halogen lamp for illumination during continuous scan to adjust before
starting the final scan. Settings of the lamp without scanning might not be effective for the image
acquisition.
•When using LSM and SIM imaging in multitrack mode, the processing of the SIM image will then lead
to a loss of the last image of the LSM acquisition. Split the images before processing the SIM image(s).

IMPORTANT NOTES
ZEISS LSM 880
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•The functions Crop, Split, Delete and Copy used in the file browser menu requires some time for GUI
update.
•When using the channel unmixing function for Airyscan data, the histogram of the resulting image still
refers to the Airyscan data set.
•Airyscan datasets with step sizes too small (much too high oversampling like with step sizes of 10 nm)
lead to faulty processing. Choose 100 nm steps or above as suggested by software for Z-Stack
acquisition.
•When analyzing PALM data for particle tracking, it is possible to select an entry in the table to
highlight the linked particle. It does not work the other way round.
•Do not use PALM image that has already been processed, again as input image for PALM processing.
This leads to GUI artefacts that are only reversible with starting ZEN again.
•When performing Z-Stacks which require averaging of frames, use Line Average. Frame Average can
lead to empty image frames.

CHAPTER 1 - SYSTEM OPERATION
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CHAPTER 1 SYSTEM OPERATION
CONTENTS
Page
1PURPOSE .............................................................................................................. 8
1.1 Software......................................................................................................................... 8
1.2 Convention for the Text in this Manual ....................................................................... 8
1.3 Backup............................................................................................................................ 9
1.4 Software Operation....................................................................................................... 9
2HARDWARE ASPECTS........................................................................................ 10
2.1 Principle of Laser Scanning Microscopy...................................................................... 10
2.2 Principle of Fluorescence Correlation Spectroscopy................................................... 11
2.3 Principle of Airyscanning............................................................................................. 12
2.4 Optical Diagram of the LSM 880 (Schematic) ............................................................. 13
2.5 Performance Features of the LSM 880........................................................................ 14
2.6 Microscope Equipment of the LSM 880 ...................................................................... 15
2.7 Computer Hardware and Software ............................................................................ 17
3STARTUP AND SHUTDOWN OF THE SYSTEM .................................................. 18
3.1 Startup of the System.................................................................................................. 18
3.1.1 The Power Remote Switch .............................................................................................. 18
3.1.2 Starting ZEN ................................................................................................................... 20
3.2 Shutdown Procedure................................................................................................... 23
3.2.1 Exiting ZEN Software ...................................................................................................... 23
3.2.2 Switching System Power Off ........................................................................................... 25
4INTRODUCTION TO THE SOFTWARE APPLICATION LAYOUT.......................... 26
4.1 Overview on the Screen Layout.................................................................................. 26
4.2 Introduction to ZEN ..................................................................................................... 27
4.3 Function Elements ....................................................................................................... 30
4.4 Application Bar ............................................................................................................ 32
4.5 Menu Bar...................................................................................................................... 32
4.5.1 File................................................................................................................................. 34
4.5.2 Maintain ........................................................................................................................ 35
4.5.2.1 Set Spline ....................................................................................................................... 35
4.5.2.2 Camera.......................................................................................................................... 36
4.5.2.3 Hardware Administrator.................................................................................................. 36
4.5.2.4 Test Grid ........................................................................................................................ 36

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4.5.3 Help – About ..................................................................................................................37
4.6 Main Toolbar ................................................................................................................38
4.7 Status Bar......................................................................................................................40
4.8 Left Tool Area ...............................................................................................................41
4.8.1 Tool Groups and Tools.....................................................................................................43
4.8.2 Context Menu of the Left Tool Area.................................................................................44
4.9 Center Screen Area.......................................................................................................45
4.9.1 Overview of the Center Screen Area ................................................................................45
4.9.2 Context Menu of the Center Screen Area.........................................................................47
4.10 Right Tool Area.............................................................................................................48
5LEFT TOOL AREA AND HARDWARE CONTROL TOOLS.....................................50
5.1 Locate Tab.....................................................................................................................51
5.1.1 Microscope Control Tool for Axio Observer.Z1 .................................................................53
5.1.2 Microscope Control Tool for Axio Imager.Z2 ....................................................................60
5.1.3 Controls for Axio Examiner ..............................................................................................63
5.1.4 Incubation Tool ...............................................................................................................64
5.2 Acquisition Tab.............................................................................................................65
5.2.1 Configuration Functions ..................................................................................................66
5.2.2 Smart Setup ....................................................................................................................68
5.2.3 Action Buttons ................................................................................................................73
5.2.4 Multidimensional Acquisition Settings ..............................................................................75
5.2.5 Setup Manager – Laser ....................................................................................................77
5.2.6 Setup Manager – Experiment Designer.............................................................................80
5.2.7 Setup Manager – Imaging Setup......................................................................................84
5.2.7.1 Imaging Mode and Track Definition .................................................................................84
5.2.7.2 Filter Cube Selection........................................................................................................86
5.2.7.3 Objective Selection ..........................................................................................................86
5.2.7.4 Stage Control..................................................................................................................87
5.2.7.5Focus Control..................................................................................................................88
5.2.7.6 HAL Control....................................................................................................................88
5.2.7.7 X-Cite Control.................................................................................................................89
5.2.7.8 Aperture Control.............................................................................................................89
5.2.7.9 Ratio Imaging..................................................................................................................90
5.2.7.10 Laser Control ..................................................................................................................90
5.2.7.11 Control Elements of the Imaging Setup Tool ....................................................................91
5.2.7.12 Setup Imaging with Airyscan Detector..............................................................................99
5.2.8 Acquisition Parameter – Acquisition Mode .....................................................................101
5.2.8.1 Objective Selection ........................................................................................................101
5.2.8.2 Scan Mode....................................................................................................................102
5.2.8.3 Format Selection ...........................................................................................................102
5.2.8.4 Averaging and Color Depth Selection.............................................................................103
5.2.8.5 HDR(High Dynamic Range)-Imaging ...............................................................................104
5.2.8.6 Scan Area Definition......................................................................................................105
5.2.8.7 Acquisition Mode Functions for LSM ..............................................................................106
5.2.8.8 Acquisition Mode Settings for Airyscan ..........................................................................113
5.2.9 Acquisition Parameter – Channels..................................................................................115

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5.2.9.1 Defining Channels and Tracks ....................................................................................... 116
5.2.9.2 Ratio Channels ............................................................................................................. 120
5.2.9.3 Using GaAsP Detectors and the temperature controlled side PMT for Photon Counting
Imaging........................................................................................................................ 121
5.2.9.4 Channel Settings for Airyscan ....................................................................................... 124
5.2.10 Acquisition Parameter – Focus....................................................................................... 126
5.2.11 Acquisition Parameter – Focus Devices and Strategy....................................................... 127
5.2.12 Acquisition Parameter – Stage....................................................................................... 131
5.2.13 Acquisition Parameter – Shuttle and Find....................................................................... 132
5.2.14 Acquisition Parameter – Regions ................................................................................... 138
5.2.15 Multidimensional Acquisition – Z-Stack.......................................................................... 141
5.2.15.1 Performing a Z-Stack Using First/Last Mode ................................................................... 142
5.2.15.2 Performing a Z-Stack Using Center Mode ...................................................................... 144
5.2.15.3 Performing a Z-Stack Using a Z-Piezo............................................................................. 146
5.2.15.4 Optimize Sectioning and Step ....................................................................................... 147
5.2.15.5 Correction.................................................................................................................... 148
5.2.15.6 Z-Stack Settings for Airyscan ......................................................................................... 149
5.2.16 Multidimensional Acquisition – Bleaching ...................................................................... 150
5.2.17 Multidimensional Acquisition – Tile Scan ....................................................................... 152
5.2.18 Scan Overview Image for Tiling ..................................................................................... 154
5.2.19 Multidimensional Acquisition – Time Series.................................................................... 155
5.2.19.1 Interval Time................................................................................................................. 155
5.2.19.2 Marker ......................................................................................................................... 157
5.2.19.3 Start............................................................................................................................. 159
5.2.19.4 End .............................................................................................................................. 161
5.2.20 Multidimensional Acquisition – Positions ....................................................................... 162
5.2.20.1 Position List .................................................................................................................. 162
5.2.20.2 Sample Carrier.............................................................................................................. 163
5.2.20.3 Setting Individual Offset Values for Positions in Position List Mode ................................. 166
5.2.20.4 Lowering Objective when Moving the Stage.................................................................. 166
5.2.20.5 Scan Overview Image.................................................................................................... 167
5.2.21 Multidimensional Acquisition – Information on Experiment ............................................ 167
5.2.22 Multidimensional Acquisition – Auto Save ..................................................................... 168
5.2.23 Image Optimization ...................................................................................................... 169
5.2.23.1 Requirements ............................................................................................................... 169
5.2.23.2 Defining the Scan Parameters........................................................................................ 171
5.2.23.3 Defining the Detection Parameters ................................................................................ 171
5.2.23.4 How to Optimize Image Quality .................................................................................... 172
5.2.23.5 How to Optimize Imaging with Airyscan........................................................................ 173
5.3 FCS Tab....................................................................................................................... 175
5.3.1 Setup – Laser................................................................................................................ 177
5.3.2 Setup – Light Path ........................................................................................................ 177
5.3.2.1 Light Path for 2 Channel Module (BiG.2 on DC out port) ............................................... 179
5.3.2.2 NDD Light Path (BiG.2 as NDD) ..................................................................................... 181
5.3.2.3 Light Path for 32 Channel Module ................................................................................ 183
5.3.2.4 Selection of Laser Lines ................................................................................................. 184
5.3.2.5 Pinhole Adjustment ...................................................................................................... 186
5.3.3 Setup – Acquisition....................................................................................................... 190

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5.3.4 Setup – Fit.....................................................................................................................192
5.3.4.1 Defining a New Model...................................................................................................194
5.3.4.2 Correlation Options.......................................................................................................195
5.3.4.3 Model Equations ...........................................................................................................196
5.3.4.4 Definition of a Model ....................................................................................................213
5.3.4.5 PCH..............................................................................................................................222
5.3.4.6 Formula ........................................................................................................................223
5.3.5 Setup – Correlator.........................................................................................................224
5.3.6 Navigation – Stage ........................................................................................................228
5.3.7 Navigation – Sample Carrier...........................................................................................229
5.3.8 Navigation – Focus ........................................................................................................231
5.3.9 Multidimensional Acquisition Tools – Time Series............................................................236
5.3.10 Multidimensional Acquisition Tools – Positions ...............................................................238
5.3.11 Navigation – Incubation.................................................................................................247
5.3.12 Multidimensional Acquisition Tools – Information on Experiment ....................................248
5.3.13 Action Buttons for FCS ..................................................................................................249
5.3.14 Method Tool .................................................................................................................250
5.3.15 Activation Tab Area.......................................................................................................253
5.3.16 New..............................................................................................................................253
5.3.17 Count Rate ...................................................................................................................254
5.3.18 X-Y-Z Scan ....................................................................................................................256
5.3.19 Snap .............................................................................................................................259
5.3.20 Start Experiment............................................................................................................259
5.3.21 Taking FCS Measurements.............................................................................................260
5.3.21.1 Start and End of a Measurement ...................................................................................263
5.3.21.2 Loading Stored Data Files ..............................................................................................267
5.3.21.3 FCS Correlation Diagram ...............................................................................................268
5.3.21.4 Image View Controls – Correlation Tab ..........................................................................271
5.3.21.5 Image View Controls – Table Tab...................................................................................273
5.3.21.6 Saving Correlation Results .............................................................................................278
5.3.21.7 Coincidence ..................................................................................................................280
5.3.21.8 Fit.................................................................................................................................281
5.3.21.9 Print..............................................................................................................................290
5.3.21.10 Information...................................................................................................................292
5.4 Processing Tab ............................................................................................................293
5.4.1 General Structure of the Processing Tab.........................................................................293
5.4.2 Processing – Airyscan Processing....................................................................................297
5.4.3 Processing – Maximum Intensity Projection.....................................................................300
5.4.4 Processing – Color-coded Projection...............................................................................301
5.4.5 Processing – Image Calculator........................................................................................302
5.4.6 Processing – Average.....................................................................................................304
5.4.7 Processing – Filter..........................................................................................................305
5.4.8 Processing – Linear Unmixing.........................................................................................308
5.4.9 Processing – Ion Concentration......................................................................................313
5.4.9.1 Single Wavelength Dyes – Offline Calibration .................................................................315
5.4.9.2 Ratiometric Dyes ...........................................................................................................316
5.4.10 Processing – Correlation ................................................................................................319
5.4.11 Processing – Modify Series.............................................................................................320

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5.4.12 Processing – HDR-imaging ............................................................................................ 321
5.4.13 Processing – Stitch........................................................................................................ 325
5.4.14 Processing – PALM........................................................................................................ 326
5.4.14.1 PALM – Convert to Image............................................................................................. 327
5.4.14.2 PALM – Import PALM Molecules ................................................................................... 328
5.4.14.3 PALM – Localization Precision........................................................................................ 329
5.4.14.4 PALM – PALM .............................................................................................................. 330
5.4.14.5 PALM – Remove PALM Outliers..................................................................................... 338
5.4.15 Processing – Particle Tracking........................................................................................ 339
5.4.16 Processing – Experimental PSF....................................................................................... 341
5.4.17 Processing – Structured Illumination .............................................................................. 345
5.4.17.1 Structured Illumination – Fourier Transform ................................................................... 346
5.4.17.2 Structured Illumination – Structured Illumination............................................................ 347
5.4.17.3 Structured Illumination – TIRF-Filtered............................................................................ 352
5.4.18 Processing – Channel Alignment ................................................................................... 354
5.4.19 Processing – Lightsheet Processing ................................................................................ 361
5.4.19.1 Dual Side Fusion ........................................................................................................... 361
5.4.19.2 Multiview Processing .................................................................................................... 364
5.4.19.3 Online Multiview Processing.......................................................................................... 381
5.4.20 Processing – Copy......................................................................................................... 382
5.4.21 Processing – ICS ........................................................................................................... 384
5.4.21.1 Detrend........................................................................................................................ 384
5.4.21.2 Remove Structure ......................................................................................................... 385
5.4.21.3 ICS Correlation ............................................................................................................. 386
5.4.21.4 Map Filter..................................................................................................................... 387
5.4.21.5 NandB Analysis............................................................................................................. 388
5.4.22 Processing – Adjust....................................................................................................... 391
5.4.22.1 Burn in Brightness and Contrast.................................................................................... 391
5.4.22.2 Interpolate Brightness and Contrast .............................................................................. 392
5.4.22.3 Channel Shift ............................................................................................................... 393
5.4.22.4 Shading Correction....................................................................................................... 394
5.5 Maintain Tab
.............................................................................................................. 395
5.5.1 Maintain – Adjust Pinhole and Collimators .................................................................... 396
5.5.1.1 Pinholes Section Panel .................................................................................................. 397
5.5.1.2 Collimators Section Panel.............................................................................................. 398
5.5.1.3 How to Adjust the Pinhole and Collimator..................................................................... 399
5.5.2 Maintain – Objectives ................................................................................................... 402
5.5.2.1 Objective...................................................................................................................... 402
5.5.2.2 Select Objective ............................................................................................................ 403
5.5.2.3 Add Objective............................................................................................................... 404
5.5.2.4 Remove Objective......................................................................................................... 405
5.5.2.5 Show/Edit Objective...................................................................................................... 405
5.5.2.6 Focus Speed ................................................................................................................. 406
5.5.2.7 Parfocal Correction....................................................................................................... 406
5.5.3 Maintain – Filters .......................................................................................................... 407
5.5.4 Maintain – Airyscan ...................................................................................................... 408
5.5.5 Options – System Options............................................................................................. 409
5.5.5.1 System Options – Load Configurations .......................................................................... 410
5.5.5.2 System Options – Reuse................................................................................................ 410

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5.5.5.3 System Options – Hardware...........................................................................................411
5.5.5.4 System Options – Image Display.....................................................................................412
5.5.6 Options – FCS Options...................................................................................................413
5.5.6.1 FCS Options – Data .......................................................................................................413
5.5.6.2 FCS Options – Measure .................................................................................................417
5.5.6.3 FCS Options – Auto Save ...............................................................................................418
5.5.7 Options – Service...........................................................................................................418
6CENTER SCREEN AREA / IMAGE CONTAINERS - DISPLAY AND IMAGE
ANALYSIS..........................................................................................................419
6.1 Structure and Functional Concept of the Center Screen Area and the Image
Display Container .......................................................................................................419
6.1.1 General Structure..........................................................................................................419
6.1.2Container Configuration with the Container Context Menu ...........................................423
6.2 2D View.......................................................................................................................424
6.2.1 Dimensions ...................................................................................................................424
6.2.2 Display..........................................................................................................................427
6.2.3 Player............................................................................................................................428
6.2.4 Graphics .......................................................................................................................429
6.2.5 Preview View ................................................................................................................432
6.3 Split View....................................................................................................................433
6.4 Gallery View ...............................................................................................................436
6.5 Airyscan View.............................................................................................................437
6.5.1 Airyscan – View Option Tab Dimensions.........................................................................437
6.5.2 Airyscan – View Option Tab Display ...............................................................................438
6.5.3 Airyscan – View Option Tab Airyscan .............................................................................439
6.6 Ortho View .................................................................................................................443
6.6.1 Ortho – Select Function .................................................................................................444
6.6.2 Ortho – Distance Function .............................................................................................445
6.7 Cut View .....................................................................................................................446
6.8 2.5 D View...................................................................................................................447
6.9 3D View.......................................................................................................................449
6.9.1 Shadow Projection ........................................................................................................451
6.9.2 Transparency Render Mode ...........................................................................................454
6.9.3 Maximum Mode............................................................................................................455
6.9.4 Surface Render Mode....................................................................................................455
6.9.5 Mixed Render Mode......................................................................................................456
6.9.6 Clipping Planes..............................................................................................................457
6.9.7 Flying Mode ..................................................................................................................461
6.9.8 3D Rendering Settings in VisArtplus ...............................................................................461
6.9.9 Series............................................................................................................................463
6.9.10 Interactive Measurements..............................................................................................465
6.9.11 Settings ........................................................................................................................466
6.9.12 Options.........................................................................................................................467
6.9.13 3D View – Basic.............................................................................................................468
6.10 Histogram View..........................................................................................................469

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6.11 Colocalization View ................................................................................................... 472
6.11.1 How a Scatter Diagram is Generated............................................................................. 474
6.11.2 Quantitative Colocalization Parameters Shown in the Data Table.................................... 475
6.11.3 Colocalization Coefficients............................................................................................ 475
6.11.4 Overlap Coefficient, Overlap Coefficient after Manders.................................................. 476
6.12 Profile View................................................................................................................ 476
6.13 Shuttle and Find View ............................................................................................... 479
6.14 Mean of ROI: Additional View Type for Time Series................................................ 482
6.15 Kinetic / FRAP View: Additional View Type for Time Series.................................... 486
6.16 Lambda Coded: Additional View Types for Lambda Mode ..................................... 492
6.17 Unmixing View .......................................................................................................... 494
6.17.1 Automatic Component Extraction ................................................................................. 499
6.18 FRET View................................................................................................................... 501
6.18.1 Tools in the FRET View Options Control Block for Acceptor Photobleaching.................... 503
6.18.2 Tools in the FRET View Options Control Block for Sensitized Emission............................. 505
6.19 Raster Scanning Image Correlation Spectroscopy (RICS) ......................................... 508
6.19.1 Access to RICS.............................................................................................................. 509
6.19.2 Control of View Functionalities...................................................................................... 509
6.19.3 Correlation Functionalities............................................................................................. 510
6.19.4 Display Functionalities................................................................................................... 513
6.19.5 Fitting Functionalities .................................................................................................... 520
6.19.6 Model Functionalities.................................................................................................... 523
6.20 Polarization Imaging ................................................................................................. 527
6.20.1 Imaging Setup for Polarization Imaging ......................................................................... 528
6.20.2 Determination of G-factor............................................................................................. 529
6.20.3 Obtaining an Anisotropy Image..................................................................................... 530
6.21 Information View....................................................................................................... 532
7RIGHT TOOL AREA, DATA MANAGEMENT AND STORAGE........................... 534
7.1 General....................................................................................................................... 534
7.2 ZEN File Browser........................................................................................................ 535
7.2.1 Gallery View of the ZEN File Browser ............................................................................. 538
7.2.2 Form View of the ZEN File Browser................................................................................ 540
7.2.3 Table View of the ZEN File Browser ............................................................................... 541
7.3 Images and Documents Panel ................................................................................... 542
7.4 Opening of Files via the "Open" Command in the File Menu ................................. 545
7.5 Save ............................................................................................................................ 545
7.6 Export of Images........................................................................................................ 546
8INDEX ............................................................................................................... 547

CHAPTER 1 - SYSTEM OPERATION
ZEISS Purpose LSM 880
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1Purpose
This section describes the operation of the Laser Scanning Microscope with the ZEN 2 software.
When starting up and operating the microscope system, mind the operating instruction manuals for the
Axio Imager.Z2, Axio Imager.M2, Axio Observer.Z1 and Axio Examiner.Z1 microscopes:
−M70-2-0020 Axio Imager.Z2 / M2, Operating Manual
−B 40-0111 Axio Observer.Z1, Operating Manual
−M60-2-0003 Axio Examiner.Z1, Operating Manual
1.1 Software
The ZEN software is used to
−control the microscope, the scanning module, the laser module, and the image acquisition process
−display, edit and analyze the images
It is a special user interface (desktop) based on the network-capable graphic 64-bit Microsoft®
WINDOWS 7 operating system.
Portions©Copyright 2007, Microsoft Corporation. All rights reserved.
The installation of the software for the Laser Scanning Microscope and the basic settings of the
equipment components are carried out by ZEISS service staff. This job includes the creation of a
customized software configuration in line with the specific hardware components of the customer's
microscope system.
1.2 Convention for the Text in this Manual
All the originally used terms of the software interface, e.g.
−names of windows
−tool groups
−panels
−input boxes
−list / selection boxes
−check boxes
−menu items
−names of buttons and sliders
−keyboard keys
are displayed in bold letters to allow easier identification.

CHAPTER 1 - SYSTEM OPERATION
LSM 880 Purpose ZEISS
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1.3 Backup
System backup
Prior to the delivery of each system, a backup of the entire system computer is created in the factory. This
backup image belongs to the system and may be re-installed in order to restore the original state of the
system computer. Please contact your local service representative if the backup image needs to be
installed.
User files backup
The following user-generated files should be included in a backup procedure controlled and carried out
on a regular basis by the user (keep directory structure):
−Carl Zeiss Image files: *.czi
−LSM Image files: *.lsm
−Exported images: *.* (*.Tiff, *.LSM-Tiff, *.BMP, …)
−Palette files: ZEN\Palette\*.lut
The backup of further hardware calibration settings including the database and licence file can easily be
created with the CanCheck tool (C:\ZEN\CanCheck).
•Click the CREATE REPORT button in the
CanCheck window (Fig. 1).
A system report will be created and saved to the
desktop on your computer as a zip file named
report.zip. This report contains all the information
needed for a system status report and to create a
save copy in case the system has to be reset.
1.4 Software Operation
The ZEN software can be operated using the
mouse, the PC keyboard, or both. The operation of
the mouse and the keyboard is identical to that of
the Microsoft® WINDOWS 7 operating system and
is therefore not described in this manual. If
required, see the Microsoft manual or online help
for relevant information.
Fig. 1 Cut-out of the CanCheck window

CHAPTER 1 - SYSTEM OPERATION
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2Hardware Aspects
2.1 Principle of Laser Scanning Microscopy
To yield information on their inner structure by conventional transmitted-light microscopy, specimens
have to be very thin and translucent; otherwise image definition will be poor. In many cases it is a
problem to satisfy these requirements.
The essential considerations have led to trailblazing changes in conventional microscopy and supplied a
successful solution of the problem above.
−Unlike the practice of even illumination in conventional microscopy, the LSM technique projects the
light of a point light source (a laser) through a high-NA objective onto a certain object plane of
interest as a nearly diffraction-limited focus. However, if not for another "trick", the stray light
produced outside the object plane, or the fluorescence of fluorescent specimens, would disturb the
in-focus image of object point of interest, resulting in a blurred image of poor contrast. The
problem therefore is how to capture only the light coming immediately from the object point in
focus, while obstructing the light coming from out-of-focus areas of the specimen.
−The light reflected, or the fluorescence light
produced, at the focus of the high
NA-objective is projected onto a variable
pinhole diaphragm by the same objective
and a tube lens. The focus inside the
specimen and the pinhole are situated at
optically conjugate points (Confocal
imaging). The decisive advantage of this
arrangement is the fact that essentially no
other light than that coming from the object
plane of interest can pass the narrow pinhole
and be registered by a detector. Unwanted
light coming from other specimen areas is
focused outside the pinhole, which passes
only a small fraction of it. The smaller the
pinhole, the less stray light or fluorescence
from out-of-focus areas will get on the
detector. The image point thus generated is
largely free from blur caused by unwanted
light.
−In order to obtain an image of the selected
object plane as a whole, it is necessary to
scan the object plane in a point-by-point,
line-by-line raster by means of an XY light
deflection system. The detectors - as a rule, photomultipliers - convert the optical information into
electric signals. This allows the image of any object plane to be generated and stored within less
than a second. By a defined focusing (Z axis) movement it is possible to look at any object plane of
interest. By scanning a succession of object planes in a specimen, a stack of slice images can be
produced.
This way, the LSM technique in conjunction with ICS optics (Infinity Color-Corrected System) has brought
decisive improvements over conventional microscopy in terms of resolving power and confocal depth contrast:
Object features in the order of 0.2 µm can be resolved, and height differences of less than 0.1 µm made
visible, without the use of interference methods.
Fig. 2 Principle of confocal imaging

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2.2 Principle of Fluorescence Correlation Spectroscopy
Fluorescence Correlation Spectroscopy (FCS) analyzes the diffusion time of molecules and their
differences if they have bound together. This is done by fluctuation analysis of fluorescence-labeled
molecules within a well defined volume element. In most experiments, Brownian motion drives the
fluctuation. The volume element is the confocal volume defined by the excitation spot of a well focused
laser beam and the selected emission region defined by the properly aligned pinhole of the detection
optics (Fig. 2).
As such, the setup is the same as for a Laser-Scanning Microscope (LSM), but in the latter we are not
interested in the fluctuations but in the average intensity. As a matter of fact, what is the signal in FCS is
noise in the LSM. Since the fluctuations are more pronounced, if fewer molecules are in the volume, FCS
requires little molecule numbers (1-10). Whereas LSM is a scanning technique, FCS uses the beam is
parked in one spot.
The fluctuations are analyzed by treating the measured photon counts with mathematical methods called
correlation functions (Fig. 3). The amplitude of the function is inverse proportional to the molecule
number and the decay time gives the residence time of the molecule in the confocal volume and hence
its diffusion time. If the two interacting molecules are of different size, only the smaller one has to be
labeled using fluorescent dyes. This method is called auto-correlation. In this case the total auto-
correlation is the sum of the two different species. If the diffusion constants of both partners are similar,
they are both labeled with different dyes and cross-correlation is used. Often, photo physical processes
like triplet states impinge on the correlation function, but can be accommodated in the model. Then the
total correlation is the product of the single processes.
Fig. 3 shows a correlation curve for a two component translational diffusion with triplet. The two
components can be separated on behalf of their diffusion time (circles). Note that the contribution of the
two components adds up, whereas the contribution of the total diffusion process and the triplet multiply
to obtain the total correlation.
Because of the tiny size of the confocal volume and its nature, the measurement can be carried out, in
principle, in every area that is reachable by light and that is not smaller than an Escherichia coli bacterium
(approximately 0.2 fl). In particular, measurements can be done inside living cells or on cell membranes.
In order to be able to place the measurement volume at its proper place, it is advantageous to combine
FCS with powerful light microscopy, particularly a confocal LSM.
Fig. 3 Correlation functions

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2.3 Principle of Airyscanning
The Airyscan detector consists of 32 elements based on GaAsP PMT technology with each element
detecting the emission signal of a focal spot much smaller than the typical 1 Airy volume.
This detection scheme together with adapted deconvolution algorigthms allows resolving fluorescently
labelled structures to a much higher degree. The resolution reaches 140 nm in x and y and 400 nm along
the optical axis.
The emission signal is filtered using standard emission filters.
The detected signal is processed online for immeditate judgement of the resulting 2D image. As the
detector works along the confocal principle an increase in resolution is achieved in 3D and using standard
specimens prepared for confocal imaging.
Another detection mode provides the ability to virtually open or close the pinhole after the image has
been acquired. In addition the detector can be used as an additional confocal detector and in
combination with other detectors of the system.

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2.4 Optical Diagram of the LSM 880 (Schematic)
AS Airyscan detector
MBS Main Beam Splitter
DBS Dichroic Beam Splitter
NDD Non-Descanned Detector
EF Emission Filter
PMT Photomultiplier
HAL Halogen Lamp
T-PMT Transmission-Photomultiplier
HBO Mercury Vapor Short-Arc Lamp
ZO Zoom Optics
IBF Infrared Blocking Filter
GaAsP Gallium Arsenide Phosphide
Fig. 4 Optical path on an inverted microscope, schematic (2 PMT / 32 array configuration)
The diagram above is a schematic representation of the LSM 880 system with attached Airyscan and
BiG.2 module.
Laser light is focused onto the specimen through an objective in a diffraction-limited mode. Light emitted
at the focal plane and at planes below and above it is directed via an XY scanner onto a main dichroic
beam splitter (MDBS), which separates the emissions from the excitation light. In the LSM 880 the
fluorescence is either spectrally separated by passing through a grating onto the Quasar detector (3 PMTs
or 2 PMTs plus 1 central GaAsP detector or 2 PMTs plus 32 channel GaAsP array) or directed via
secondary beam splitters into Airyscan or BiG.2 module. In Airyscan light is filtered by emission filters
(EF5) and projected onto the Airyscan detector with its 32 sub-airy detector elements. In the BiG.2
module the light passes through a Push & Click filter cube with a dichroic beam splitter (DBS 6) and two
emission filters (EF6 and EF7) onto 2 GaAsP (GaAsP1 and GaAsP2) detectors. The pinhole (VP) blocks all
out of focus light, whereas emission filters (EF) are used to select a specific spectrum of the emitted light
and to suppress any remaining excitation light. In NLO applications, light can be either directed to the
Quasar detector or BiG.2 module (descanned detection) or to NDDs (equipped with either multialkali or
GaAsP PMT) in transmission or reflection position (non-descanned detection).

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2.5 Performance Features of the LSM 880
Optical and Mechanical Aspects
The highly integrated system design makes for the shortest possible optical paths, top-grade optical
precision and high stability. The Airyscan module can be mounted to the LSM 880 scan head of any
configuration and with upright or inververted microscopes. The BiG.2 module is either directly attached
to the scan head and can be used in combination with upright and inverse microscope stands.
The spectral range available extends from the UV to the IR region.
For the VIS (visible-light) Laser Module, the user can select from up to six lasers with wavelengths of 633,
594, 561, 543, 514, 488, 458 and 405 nm. The UV Laser Module provides wavelengths of 355 nm. Ti:Sa
Laser provide pulsed laser light from 680 to 1300 nm for Multiphoton imaging (NLO). UV and visible
lasers are coupled to the scan head via polarization-preserving single-mode optical fibers. Ti:Sa lasers are
coupled to the sytem via free beam coupling kits. One variable beam collimator each for the UV, NLO and
visible ranges provides optimum adaptation of the respective laser wavelength to the objective used and,
thus, optimum correction for Z aberrations.
Acousto-optical tunable filters (AOTF) adjust the necessary brightness for up to 8 laser lines within
microseconds. Ti:Sa lasers are attenuated via AOM within less than a microsecond.
The internal image acquisition channels usable for reflection or fluorescence, and an additional
transmitted-light channel are ideal for the investigation of multiple fluorescence specimens. Airyscan and
BiG.2 are attached directly to the scan head. Both are equipped with Gallenium Arsenide Phosphide
detectors (GaAsP). The diameter of the pinhole and the XY positions can be optimized, and the desired
emission can be selected. This adjustment also includes positioning along Z. For the simultaneous
registration of multiple fluorescence signals, identical optical sections can be obtained in each confocal
channel.
The microscope's transmitted-light channel is equipped with a photomultiplier, too. It is therefore
possible to superimpose a multiple fluorescence image on a brightfield, differential interference or phase
image.
Airyscan is equipped with a filter wheel consisting of 8 positions of which 6 are used for specific double
band pass emission filters. BiG.2 dichroics and emission filters are housed in push & click filter cubes
which are exchanged manually.
The high-NA C-APOCHROMAT objectives specially developed for the LSM technique reach the physical
limit in resolving power, and can be used throughout the 380...900 nm spectral range with the same
high quality, producing brilliant images. Note, for fluorescence correlation spectroscopy the
C-APOCHROMAT objectives are highly recommended.
A two-mirror scanner system, controlled by real time electronics, offers several advantages. The large
deflection angle of the scanning mirrors allows a wide area to be scanned. With a 1.25×objective, the
object area scanned is 11 ×11 mm². The scanning field size can be freely selected between 4 ×1 and
8192 ×8192 pixels.
It is possible to rotate the XY scanning field through 360° and carry out XY scans without having to
rotate the specimen itself under laser radiation load.
For FCS operation all detectors able to count photons can be used for FCS. These are GaAsP PMTs of the
array detector, BiG.2 and the cooled multialkali PMT in the red spectral range. Multialkali PMTs of the
three channel configuration without GaAsP PMTs or NDDs cannot be used for FCS.
Selection of the specimen detail of interest for zooming is fast and convenient, and the zoomed image is
automatically centered. This saves the job of specimen centration with the microscope stage.
Bi-directional scanning doubles the scanning rate to a maximum of 13 frames/sec (at 512 ×512 pixels).
Two fluorescent dyes can be viewed and documented in a quasi-simultaneous mode when using two
different laser wavelengths for bi-directional scanning. This will prevent cross talk between detection
channels.