Zeiss ELYRA 7 User manual

ELYRA
7
Operating
Manual
March 2019
ZEN 3.0 SR (black edition)

ZEISS Copyright ELYRA 7
II 000000-2262-999 03/2019 V_02
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
ELYRA 7 ZEISS
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How to make best use of the ELYRA 7 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 ELYRA 7
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Contents
1
System Operation
In this section you will find the most important steps and procedures of the 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.

CHAPTER 1 - SYSTEM OPERATION
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CHAPTER 1 SYSTEM OPERATION
CONTENTS
Page
1PURPOSE .............................................................................................................. 7
1.1 Software......................................................................................................................... 7
1.2 Convention for the Text in this Manual ....................................................................... 7
1.3 Backup............................................................................................................................ 8
1.4 Software Operation....................................................................................................... 8
2HARDWARE ASPECTS.......................................................................................... 9
2.1 Principles of Superresolution Microscopy (ELYRA) ...................................................... 9
2.1.1 Principle of Structured Illumination Microscopy (SIM) ......................................................... 9
2.1.2 Principle of Photo Activated Localization Microscopy (SMLM) ........................................... 11
2.2 Optical Diagram of ELYRA (Schematic)....................................................................... 13
2.3 Performance and Features of ELYRA .......................................................................... 15
2.4 Microscope Equipment of ELYRA Systems ................................................................. 16
2.5 Computer Hardware and Software ............................................................................ 18
3STARTUP AND SHUTDOWN OF THE SYSTEM .................................................. 19
3.1 Startup of the System.................................................................................................. 19
3.1.1 Starting ZEN ................................................................................................................... 20
3.2 Shutdown Procedure................................................................................................... 23
3.2.1 Exiting ZEN Software ...................................................................................................... 23
3.2.2 Switching System Power Off ........................................................................................... 24
4INTRODUCTION TO THE SOFTWARE APPLICATION LAYOUT.......................... 25
4.1 Overview on the Screen Layout.................................................................................. 25
4.2 Introduction to ZEN ..................................................................................................... 26
4.3 Function Elements ....................................................................................................... 29
4.4 Application Bar ............................................................................................................ 31
4.5 Menu Bar...................................................................................................................... 31
4.5.1 File................................................................................................................................. 33
4.5.2 Maintain ........................................................................................................................ 34
4.5.2.1 Camera.......................................................................................................................... 34
4.5.2.2 Hardware Administrator.................................................................................................. 34
4.5.2.3 Test Grid ........................................................................................................................ 34
4.5.2.4 Remote Access Settings .................................................................................................. 35
4.5.3 Help – About.................................................................................................................. 36

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4.6 Main Toolbar ................................................................................................................37
4.7 Status Bar......................................................................................................................39
4.8 Left Tool Area...............................................................................................................40
4.8.1 Tool Groups and Tools.....................................................................................................42
4.8.2 Context Menu of the Left Tool Area.................................................................................43
4.9 Center Screen Area.......................................................................................................44
4.9.1 Overview of the Center Screen Area ................................................................................44
4.9.2 Context Menu of the Center Screen Area.........................................................................46
4.10 Right Tool Area.............................................................................................................47
5LEFT TOOL AREA AND HARDWARE CONTROL TOOLS.....................................49
5.1 Locate Tab.....................................................................................................................50
5.1.1 Microscope Control Tool for Axio Observer ......................................................................52
5.1.2 Incubation Tool ...............................................................................................................58
5.2 Acquisition Tab.............................................................................................................59
5.2.1 Configuration Functions ..................................................................................................60
5.2.2 Action Buttons ................................................................................................................62
5.2.3 Multidimensional Acquisition Settings ..............................................................................64
5.2.4 Setup Manager – Laser ....................................................................................................66
5.2.5 Setup Manager – Imaging Setup......................................................................................67
5.2.5.1 Sequential Laser Switching ..............................................................................................68
5.2.5.2 Beam Path Configuration ................................................................................................71
5.2.5.3 Camera Selection ............................................................................................................73
5.2.5.4 Tube Lens Selection.........................................................................................................75
5.2.5.5 Filter Cube Selection........................................................................................................75
5.2.5.6 Objective Selection ..........................................................................................................75
5.2.5.7 HAL Control....................................................................................................................76
5.2.5.8 Epi Illumination Control...................................................................................................76
5.2.5.9 Aperture Control.............................................................................................................76
5.2.5.10 Power Density Control.....................................................................................................77
5.2.5.11 Grating Selection.............................................................................................................77
5.2.5.12 Collimator Setting ...........................................................................................................78
5.2.5.13 TIRF Angle Setting...........................................................................................................78
5.2.5.14 3D-PALM Slider...............................................................................................................81
5.2.6 Acquisition Parameter – Experiment Designer...................................................................82
5.2.7 Acquisition Parameter – Acquisition Mode .......................................................................85
5.2.7.1 Objective Selection ..........................................................................................................85
5.2.7.2 Format and Trigger Selection ...........................................................................................86
5.2.7.3 Averaging and Color Depth Selection...............................................................................87
5.2.8 Acquisition Parameter – Incubation..................................................................................87
5.2.9 Acquisition Parameter – Online Processing Options...........................................................88
5.2.9.1 Online Processing SMLM .................................................................................................88
5.2.9.2 Control Activation Power.................................................................................................89
5.2.10 Acquisition Parameter – Channels....................................................................................90
5.2.10.1 Defining Channels and Tracks..........................................................................................92
5.2.10.2 Ratio Channels................................................................................................................96
5.2.10.3 Track Control ..................................................................................................................97
5.2.10.4 Display Options ...............................................................................................................99

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5.2.11 Acquisition Parameter – Focus......................................................................................... 99
5.2.12 Acquisition Parameter – Definite Focus .......................................................................... 101
5.2.13 Focus Devices and Strategies for Widefield Imaging Modes............................................ 111
5.2.14 Acquisition Parameter – Stage....................................................................................... 112
5.2.15 Acquisition Parameter – Shuttle and Find....................................................................... 113
5.2.16 Acquisition Parameter – Regions ................................................................................... 120
5.2.17 Multidimensional Acquisition – Z-Stack.......................................................................... 124
5.2.17.1 Performing a Z-Stack Using First/Last Mode ................................................................... 125
5.2.17.2 Performing a Z-Stack Using Center Mode ...................................................................... 127
5.2.17.3 Performing a Z-Stack Using a Z-Piezo............................................................................. 129
5.2.17.4 Correction.................................................................................................................... 130
5.2.18 Multidimensional Acquisition – Tile Scan ....................................................................... 130
5.2.19 Scan Overview Image for Tiling ..................................................................................... 133
5.2.20 Multidimensional Acquisition – Time Series.................................................................... 134
5.2.20.1 Interval Time................................................................................................................. 135
5.2.20.2 Marker ......................................................................................................................... 137
5.2.20.3 Start............................................................................................................................. 139
5.2.20.4 End .............................................................................................................................. 141
5.2.21 Multidimensional Acquisition – Positions ....................................................................... 142
5.2.21.1 Position List .................................................................................................................. 142
5.2.21.2 Sample Carrier.............................................................................................................. 143
5.2.21.3 Lowering Objective when Moving the Stage.................................................................. 146
5.2.21.4 Scan Overview Image.................................................................................................... 146
5.2.22 Multidimensional Acquisition – Information on Experiment ............................................ 147
5.2.23 Multidimensional Acquisition – Streaming and Auto Save .............................................. 147
5.2.23.1 Streaming tool.............................................................................................................. 148
5.2.23.2 Auto Save tool.............................................................................................................. 150
5.3 Processing Tab ........................................................................................................... 152
5.3.1 General Structure of the Processing Tab ........................................................................ 152
5.3.2 Processing – Airyscan Processing ................................................................................... 156
5.3.3 Processing – Maximum Intensity Projection.................................................................... 160
5.3.4 Processing – Color-coded projection.............................................................................. 161
5.3.5 Processing – Image Calculator....................................................................................... 162
5.3.6 Processing – Average .................................................................................................... 164
5.3.7 Processing – Filter ......................................................................................................... 165
5.3.8 Processing – Linear Unmixing ........................................................................................ 168
5.3.9 Processing – Ion Concentration ..................................................................................... 173
5.3.9.1 Single Wavelength Dyes – Offline Calibration ................................................................ 175
5.3.9.2 Ratiometric Dyes........................................................................................................... 176
5.3.10 Processing – Correlation................................................................................................ 179
5.3.11 Processing – Modify Series ............................................................................................ 180
5.3.12 Processing – HDR-imaging ............................................................................................ 181
5.3.13 Processing – Stitch........................................................................................................ 185
5.3.14 Processing – Localization Microscopy Tools.................................................................... 186
5.3.14.1 Localization Microscopy Tools – Convert to Image ......................................................... 187
5.3.14.2 Localization Microscopy Tools – Import SMLM Molecules............................................... 188
5.3.14.3 Localization Microscopy Tools – Localization Precision.................................................... 189
5.3.14.4 Localization Microscopy Tools – Remove SMLM Outliers................................................. 190

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5.3.15 Processing – Localization Microscopy .............................................................................191
5.3.16 Processing – Particle Tracking.........................................................................................199
5.3.17 Processing – Experimental PSF........................................................................................201
5.3.18 Processing – SIM Tools...................................................................................................205
5.3.18.1 SIM – Fourier Transform ................................................................................................206
5.3.18.2 SIM – Modulation contrast ............................................................................................207
5.3.18.3 SIM – Stripe SIM............................................................................................................210
5.3.18.4 SIM – TIRF-Filtered.........................................................................................................215
5.3.19 Processing – SIM ...........................................................................................................217
5.3.19.1 Processing with Default setting......................................................................................218
5.3.19.2 Processing with Adjust settings ......................................................................................221
5.3.20 Processing – Channel Alignment....................................................................................223
5.3.21 Processing – Lightsheet Processing.................................................................................230
5.3.21.1 Dual Side Fusion............................................................................................................230
5.3.21.2 Multiview Processing .....................................................................................................233
5.3.21.3 Online Multiview Processing ..........................................................................................250
5.3.22 Processing – Copy .........................................................................................................251
5.3.23 Processing – ICS ............................................................................................................253
5.3.23.1 Detrend ........................................................................................................................253
5.3.23.2 Remove Structure..........................................................................................................254
5.3.23.3 ICS Correlation..............................................................................................................255
5.3.23.4 Map Filter .....................................................................................................................256
5.3.23.5 NandB Analysis..............................................................................................................257
5.3.24 Processing – Adjust .......................................................................................................260
5.3.24.1 Burn in Brightness and Contrast.....................................................................................260
5.3.24.2 Interpolate Brightness and Contrast ...............................................................................261
5.3.24.3 Channel Shift ................................................................................................................262
5.3.24.4 Shading Correction .......................................................................................................263
5.3.25 Processing – Detrending ................................................................................................264
5.4 Maintain Tab ..............................................................................................................265
5.4.1 Maintain Toul group – Dual Camera Alignment..............................................................266
5.4.2 Maintain tool group – Objectives ...................................................................................267
5.4.2.1 Detection Objective .......................................................................................................267
5.4.2.2 Focus Speed..................................................................................................................271
5.4.2.3 Parfocal Correction........................................................................................................271
5.4.3 Maintain tool group – Adjust PALM Slider......................................................................273
5.4.4 Options tool group – System Options.............................................................................275
5.4.4.1 System Options – Camera..............................................................................................276
5.4.4.2 System Options – Elyra ..................................................................................................277
5.4.4.3 System Options – Load Configurations...........................................................................278
5.4.4.4 System Options – Reuse.................................................................................................278
5.4.4.5 System Options – Hardware...........................................................................................279
5.4.4.6 System Options – Image Display.....................................................................................279
5.4.4.7 System Options – Streaming ..........................................................................................280
5.4.5 Options tool group – Experiment Designer .....................................................................280
5.4.6 Options tool group – Service..........................................................................................280

CHAPTER 1 - SYSTEM OPERATION
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6CENTER SCREEN AREA / IMAGE CONTAINERS - DISPLAY AND IMAGE
ANALYSIS ......................................................................................................... 281
6.1 Structure and Functional Concept of the Center Screen Area and the
Image Display Container ........................................................................................... 281
6.1.1 General Structure ......................................................................................................... 281
6.1.2 Container Configuration with the Container Context Menu........................................... 285
6.2 2D View...................................................................................................................... 286
6.2.1 Dimensions................................................................................................................... 286
6.2.2 Display ......................................................................................................................... 289
6.2.3 Player........................................................................................................................... 290
6.2.4 Graphics....................................................................................................................... 291
6.2.5 Preview View................................................................................................................ 294
6.3 Split View................................................................................................................... 295
6.4 Gallery View............................................................................................................... 298
6.5 Ortho View................................................................................................................. 300
6.5.1 Ortho - Select Function ................................................................................................. 301
6.5.2 Ortho - Distance Function ............................................................................................. 302
6.6 Cut View..................................................................................................................... 303
6.7 2.5 D View.................................................................................................................. 304
6.8 3D View...................................................................................................................... 306
6.8.1 Shadow Projection........................................................................................................ 308
6.8.2 Transparency Render Mode........................................................................................... 311
6.8.3 Maximum Mode........................................................................................................... 312
6.8.4 Surface Render Mode ................................................................................................... 312
6.8.5 Mixed Render Mode ..................................................................................................... 313
6.8.6 Clipping Planes............................................................................................................. 314
6.8.7 Flying Mode ................................................................................................................. 318
6.8.8 3D Rendering Settings in VisArt..................................................................................... 318
6.8.9 Series ........................................................................................................................... 320
6.8.10 Interactive Measurements ............................................................................................. 322
6.8.11 Settings........................................................................................................................ 323
6.8.12 Options ........................................................................................................................ 324
6.8.13 3D View – Basic............................................................................................................ 325
6.9 Histogram View ......................................................................................................... 326
6.10 Colocalization View ................................................................................................... 329
6.10.1 How a Scatter Diagram is Generated............................................................................. 331
6.10.2 Quantitative Colocalization Parameters Shown in the Data Table.................................... 332
6.10.3 Colocalization Coefficients............................................................................................ 332
6.10.4 Overlap Coefficient, Overlap Coefficient after Manders.................................................. 333
6.11 Profile View................................................................................................................ 333
6.12 Shuttle and Find View ............................................................................................... 336
6.13 Mean of ROI: Additional View Type for Time Series................................................ 339
6.14 Kinetic / FRAP View: Additional View Type for Time Series.................................... 343
6.15 Lambda Coded: Additional View Types for Lambda Mode ..................................... 349

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6.16 Unmixing View...........................................................................................................350
6.16.1 Automatic Component Extraction..................................................................................353
6.17 FRET View ...................................................................................................................354
6.17.1 Tools in the FRET View Options Control Block for Acceptor Photobleaching ....................356
6.17.2 Tools in the FRET View Options Control Block for Sensitized Emission .............................358
6.18 SMLM View.................................................................................................................361
6.18.1 Dimensions View Control Tab........................................................................................362
6.18.2 Display View Control Tab...............................................................................................367
6.18.3 Graphics View Control Tab ............................................................................................368
6.18.4 SMLM-Drift View Control Tab........................................................................................369
6.18.4.1 Fiducial Based Drift Correction .......................................................................................371
6.18.4.2 Model-based Drift Correction ........................................................................................378
6.18.5 SMLM-Grouping View Control Tab ................................................................................383
6.18.6 SMLM-Statistics View Control Tab..................................................................................385
6.18.7 SMLM-Filter View Control Tab .......................................................................................392
6.18.8 SMLM-Render View Control Tab....................................................................................395
6.18.8.1 Rendering Data Sets ......................................................................................................395
6.18.8.2 Visualizing Data Sets .....................................................................................................402
6.19 Particle Tracking View................................................................................................406
6.19.1 Particle Tracking Control View Tab.................................................................................406
6.20 Polarization Imaging ..................................................................................................408
6.21 Information View .......................................................................................................409
7RIGHT TOOL AREA, DATA MANAGEMENT AND STORAGE ...........................411
7.1 General........................................................................................................................411
7.2 ZEN File Browser.........................................................................................................412
7.2.1 Gallery View of the ZEN File Browser..............................................................................415
7.2.2 Form View of the ZEN File Browser.................................................................................417
7.2.3 Table View of the ZEN File Browser ................................................................................418
7.3 Images and Documents Panel....................................................................................419
7.4 Opening of Files via the "Open" Command in the File Menu..................................422
7.5 Save.............................................................................................................................422
7.6 Export of Images ........................................................................................................423
8INDEX ................................................................................................................424