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
03/2019 V_02 000000-2262-999 III
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
IV 000000-2262-999 03/2019 V_02
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
ELYRA 7 Contents ZEISS
03/2019 V_02 000000-2262-999 1
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

CHAPTER 1 - SYSTEM OPERATION
ZEISS Contents ELYRA 7
2 000000-2262-999 03/2019 V_02
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

CHAPTER 1 - SYSTEM OPERATION
ELYRA 7 Contents ZEISS
03/2019 V_02 000000-2262-999 3
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

CHAPTER 1 - SYSTEM OPERATION
ZEISS Contents ELYRA 7
4 000000-2262-999 03/2019 V_02
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
ELYRA 7 Contents ZEISS
03/2019 V_02 000000-2262-999 5
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

CHAPTER 1 - SYSTEM OPERATION
ZEISS Contents ELYRA 7
6 000000-2262-999 03/2019 V_02
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

CHAPTER 1 - SYSTEM OPERATION
ELYRA 7 Purpose ZEISS
03/2019 V_02 000000-2262-999 7
1Purpose
This section describes the operation of the Laser Scanning Microscope with the ZEN 3.0 SR software.
When starting up and operating the microscope system, mind the operating instruction manuals for the
Axio Observer7 microscope stand and microscope components:
−Axio Observer 3/5/7, Operating Manual (431004-7244-001)
−Definite Focus, Operating Manual (424533-7244-001)
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 7/10 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
ZEISS Purpose ELYRA 7
8 000000-2262-999 03/2019 V_02
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 7/10 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
ELYRA 7 Hardware Aspects ZEISS
03/2019 V_02 000000-2262-999 9
2Hardware Aspects
2.1 Principles of Superresolution Microscopy (ELYRA)
A point object imaged by an objective lens will always be a blurred spot, the so called point spread
function (PSF), due to the diffraction of light.
Therefore two points cannot come closer than a certain limit in order to still be resolved. This minimal
distance in object space corresponds to a maximal cut-off frequency in the frequency space that can be
transmitted through the objective lens.
The transmittable frequencies represent the so-called optical transfer function (OTF), which is the Fourier
transform of the PSF. As a rule of thumb the resolution of a far field light microscope in the lateral
direction is approximately half the wavelength, whereas it is 3 fold worse in the axial direction.
Hence, for a light microscope lateral and axial resolutions are approximately 200 and 600 nm,
respectively. In order to enhance resolution, the PSF has to be narrowed, which is synonymous to the OTF
to be expanded.
This exactly is accomplished by structured illumination microscopy (SIM) and photo activated localization
microscopy (SMLM).
2.1.1 Principle of Structured Illumination Microscopy (SIM)
In SIM a sinusoidal pattern, e. g. a line grid pattern (Stripe SIM), or a dot pattern (Lattice SIM) with a
defined periodicity, is positioned in the excitation path in a plane that is conjugated to the image plane
(Fig. 2).
Hence the pattern is projected onto the image and the excitation intensity is modulated along the
pattern. This results in a modulation of the fluorescence as well.
The highest modulation contrast is obtained in the focal plane. The contrast gets weaker with distance to
the focal plane so that depth discrimination in the axial direction is possible. The grid constant is ideally
the cut off frequency of the system.
The interferences of the diffraction orders generated through the grid are used for the lateral and axial
structuring of the light.
Fig. 2 Principle of structured illumination – diffraction orders from the
modulation grid (line grid pattern) and the sample structures are
indicated

CHAPTER 1 - SYSTEM OPERATION
ZEISS Hardware Aspects ELYRA 7
10 000000-2262-999 03/2019 V_02
The interference of the structured light with object
structures requires that at least part of the light is
coherent and leads to so called Moirè fringes
whose pattern has a longer periodicity and hence a
lower space frequency compared to the object
(Fig. 3).
Each sample or object structure can be regarded as
a superimposition of many grids. Due to the
structured illumination the object frequencies will
be shifted to lower frequencies due to the Moiré
effect. Therefore high frequencies that normally
cannot be collected by the system can be
transmitted due to their shift into lower
frequencies.
Due to the diffraction nature of light the
modulation grid frequency cannot have a
periodicity that is smaller than half the wavelength,
so resolution enhancement can be a maximum of
two fold in each lateral and axial direction.
Due to the frequency shift the image frequencies
are composed of non-shifted and shifted object
frequencies and hence their contribution has to be
determined.
A coherent image generated with a sinusoidal line grid pattern has generally 3 different orders created by
interference: the 0th order from the 0th order non-diffracted beam, the 1st order created by interferenes of
the ±1st order diffraction beams with the 0th order non diffracted beam (frequencies shifted by half the
modulation frequency) and the 2nd order created by the interference between the +1st order with the
– 1st order diffracted beam (frequencies shifted by the modulation frequency). (Fig. 2).
It is the 2nd order that contains the high frequency information for resolution enhancement in X,Y (Fig. 4).
The 1st order contains information for sectioning and better z-resolution.
To shift the frequencies to their correct location, a linear equation with 5 unknowns (2 x n-1, n=number
of different orders, which is 3 in the case of a line grid) has to be solved. This can be pixel wise
accomplished by recording 5 images with a phase shifted modulation pattern. Since resolution is
obtained only in the orientation of the grid pattern, the grid has to be rotated to obtain a nearly uniform
resolution in all directions. In generally one uses 3 rotations are sufficient. Hence, the reconstruction of a
Fig. 3 Moiré pattern generated by overlaying
two line grids – the lower periodicity of
the generated pattern is visible as
thicker lines
Fig. 4 Expansion of the transmitted frequencies – the cut-off frequency is
indicated by k0, phase shifts by
Φ
and rotations by
ρ
; only the shift by
the modulation frequency is shown as this contains the high
resolution information

CHAPTER 1 - SYSTEM OPERATION
ELYRA 7 Hardware Aspects ZEISS
03/2019 V_02 000000-2262-999 11
SIM image requires a minimum of 3 x 5 = 15 images for a line grid pattern. Finally a back transformation
from the frequency to the real space generates the high resolution image.
In Lattice SIM we have a 7 orders. Hence 13 (2 x 7 + 1) phase shifted images are minimally required. In
ELYRA 7 15 phase images are used to have more information for reconstruction of the SIM image. Since
the Lattice SIM pattern is symmetrical, no rotaion is necessary.
2.1.2 Principle of Photo Activated
Localization Microscopy (SMLM)
In SMLM the sample is illuminated in a regime that
statistically activates only one fluorescent molecule
per PSF.
Hence, the centre of mass of the PSF can be
determined, which can be done more precisely
than the PSF itself. The accuracy of determination
depends solely on the number of photons
collected.
The resolution enhancement is by a factor of 1/√N
with N representing the number of photons
(Fig. 5).
To image molecules one at a time in a PSF requires
certain properties of the fluorescence dye.
It has to exist in a bright and dark state, which can
be converted between each other.
Most of organic dyes show such a behavior in
certain reducing environments.
And there are also three classes of fluorescent
proteins (FPs) with the required switching
characteristics.
Photoactivatable FPs can be irreversibly switched from a dark to a bright state by violet light.
The bright state is than bleached by the imaging laser light. Photoconvertable FPs can be irreversible
switched from one spectral state to another by violet light.
The latter one is than bleached by the imaging laser.
Photoswitchable FPs are reversibly switched from a dark state to bright state by violet light and will turn
back to the ark state by illumination with the imaging laser light.
It is the correct balance between the activation light (mostly violet light) and the imaging light (mostly
strong powers needed) to that determines how many molecules are in the on-state and how many
molecules are in the off-state.
Fig. 5 The accuracy of the determination of
the center of mass depend on photon
number N

CHAPTER 1 - SYSTEM OPERATION
ZEISS Hardware Aspects ELYRA 7
12 000000-2262-999 03/2019 V_02
Since only a few molecules are active at a time the procedure of activating to convert molecules to the
on-state followed by deactivation to convert them to the off-stateh has to be repeated many times in
order to detect all labeled molecules (Fig. 6).
So the aim in SMLM is to activate a few molecules, calculate their center of mass, plot them in a new
coordinate system and deactivate them quickly (preferentially after 1 to 3 frames) and repeat the process
until statistically all molecules have been activated.
So a SMLM experiment easily needs 10 000 frames and more.
The resulting image is a plot of all molecules localizations with a certain precision depending on the
photon numbers obtained from the molecule.
Fig. 6 Principle of SMLM – Whereas in normal widefield images the PSF of
closely juxtaposed point sources can overlap heavily (upper panel)
their sequential illumination and plotting of their center of mass will
result in their clear separation (lower panel)

CHAPTER 1 - SYSTEM OPERATION
ELYRA 7 Hardware Aspects ZEISS
03/2019 V_02 000000-2262-999 13
2.2 Optical Diagram of ELYRA (Schematic)
Fig. 7 Optical path, schematic (ELYRA 7)
The diagram is a schematic representation of the ELYRA system (Fig. 7).
Laser light is focused into the back focal plane of the objective to obtain widefield illumination in the
specimen. For TIRF mode dependent on the angle of the TIRF mirror either Epi-, HILO- or TIRF-illumination
is achieved.
For SIM mode only Epi-illumination is available as the TIRF mirror remains in a fixed position. Light
emitted from the specimen is directed back via a reflector cube that separates the emission from the
excitation light. The fluorescence signals are directed to individual EMCCD cameras.

CHAPTER 1 - SYSTEM OPERATION
ZEISS Hardware Aspects ELYRA 7
14 000000-2262-999 03/2019 V_02
Attachment sites for illumination modules and cameras
The ELYRA 7 module is always attached at the rear port (RP) of the Axio Observer 7 SR. An LSM module
is always mounted to the left side port (SPl) of the stand. The Duolink will always be mounted, if present,
on the right side port (SPr).
For ELYRA 7 type L systems an Andor iXon 897 Ultra EMCCD camera is mounted on the right side port
(SPr).
For ELYRA type LS systems, the Andor iXon U897 Ultra EMCD camera is mounted to the rtght side port
(SPr), whereas the pco.edge CHL sCMOS camera will be mounted to the base port (BP).
For ELYRA type S and eLS systems the pco.edge CHL sCMOS camera will be mounted to the right side
port (SPr).

CHAPTER 1 - SYSTEM OPERATION
ELYRA 7 Hardware Aspects ZEISS
03/2019 V_02 000000-2262-999 15
2.3 Performance and Features of ELYRA
Optical and mechanical aspects
The ELYRA 7 family members have only one illumination / detection unit, the ELYRA 7 module, attached
to the rear port of the microscope.
The ELYRA module is designed for performing single molecule localization microscopy (SMLM) and/or
Lattice structured illumination microscopy (Lattice SIM) SMLM. The ELYRA 7 family consists of ELYRA type
L (SMLM only), ELYRA type S (SIM only) and ELYRA types LS and eLS (SMLM and SIM combination).
The highly integrated system design allows combining two super resolution illumination modes, SMLM
and SR-SIM in one module. The ELYRA illumination module can be fitted to an inverted Axio Observer 7
SR microscope at the rear port.
The spectral range available extends from the V to the VIS region.
For the VIS (visible-light), the user can select from up to 3 lasers with wavelengths of 641, 561 and
488 nm. For the V (violet light) a 405 nm laser is available. Coupling of the laser light is through
polarization-preserving single-mode optical fibers. One variable beam collimator provides adaptation of
the respective laser wavelength to the objective used.
Acousto-optical tunable filters (AOTF) adjust the necessary brightness for all 4 laser lines within
microseconds. The 405 laser is in addition equipped with neutral density filters that allow tuning the laser
power further down.
The implemented cameras, the pco.edge CLHS sCMOS camera for Lattice SIM & SMLM and the Andor
iXon 897 Ultra for SMLM, are optimized for the different technologies.
The high-NA Plan Apochromat 63x (NA 1.4), α-Plan Apochromat 63x (NA 1.46) and C-Apochromat 63x
(NA 1.2) objectives are especially suited for performing Lattice SIM experiments. The α-Plan Apochromat
100x (NA 1.46) and α-Plan Apochromat 100x (NA 1.57) HI objectives with their back reflection blocker
have been specifically designed for SMLM applications minimizing scatter light in TIRF mode.
For ELYRA types L, LS and eLS the TIRF mirror is moveable so the optimal TIRF angle for TIRF-illumination
can be set. In ELYRA type S systems, this mirror is in a fixed position for Epi-illumination. ELYRA types S,
LS and eLS systems have 5 grids with different grid constants to adapt laser wavelength, objective and
penetration depth in Lattice SIM illumination. Galvo mirros are used to move the grids laterally.
The field of view (FOV) in SIM is ~ 75 x 75 µm (63 x objective, 1.6x Optovar, 1280 x 1280 pixel with a
pitch of 6.45 µm) using the pco.edge CLHS sCMOS camera. The FOV in SMLM using the Andor iXon 897
Ultra with 512 x 512 pixel (16 µm x 16 µm pixel pitch) is ~ 50 x 50 µm and can be further reduced
providing higher power densities.

CHAPTER 1 - SYSTEM OPERATION
ZEISS Hardware Aspects ELYRA 7
16 000000-2262-999 03/2019 V_02
2.4 Microscope Equipment of ELYRA Systems
The ELYRA 7 systems are equipped with the inverted Axio Observer 7 SR microscope with a modified rear
port for the attachment of ELYRA illumination modules.
Only the differences from the delivered operating manual "Axio Observer" will be explained here.
(1) Stand
−The motorized objective nosepiece 5×H DIC is firmly fixed to the stand, where no operating
elements can be found for the nosepiece. Operation will be performed via ZEN software control.
The "Restriction of the nosepiece height to protect the objectives during motorized objective
change" is inactivated. The nosepiece will be moved down automatically before each motorized
objective change.
−The reflector mount is motorized and provided with the Axio Observer 7 SR reflector turret. The
reflector turret has six positions: One transmitting light position (position 1), which is empty, one or
more further positions (position 2-6) for push & click fluorescence filter sets or analyzer/polarizer
cubes (reflector module) to be used for optical observation. For ELYRA four of those positions
(positions 3-6) are adjusted and used in Lattice SIM and SMLM illumination regimes. For SMLM it is
also possible to use a push & click filter set. If you want to use more conventional fluorescence filter
sets, you need to employ a further reflector turret. When changing the reflector turret position you
must make sure that the turret will click into position, since otherwise the image area will be cut.
The reflector turrets are equipped with auto component recognition (ACR), so the actual filter sets
will be updated and displayed in the ZEN software.
−The stand has a motorized focusing drive (fine & coarse). Sensitivity of the focusing drive is adjusted
to the delivered objectives by the manufacturer but can be modified using the controls on the TFT
display of the stand. The stand features an integrated power supply for the internal motors and
stand electronics. External power supply units are used for the mercury vapor short arc lamp or the
fiber coupled lamp.
−The analyzer slider for conventional DIC methods will be operated from the right side and is located
just below the nosepiece.
−When the rod is pushed in, the analyzer is located in the beam path. In the laser widefield modes
(Lattice SIM, SIM Apotome, SMLM, TIRF), the analyzer must not be located in the beam path, and
the analyzer rod must be pulled out.
−When using SMLM, SIM Apotome and Lattice SIM, do not use a DIC slider.
(2) Specimen stages and fine focus drives
−XY-Piezo Scanning stage for motorized positioning in X and Y.
−Z-Piezo stage insert for motorized positioning in Z. The z-Piezo stage insert is obligatory for SR-SIM
illumination. It is highly recommended for SMLM illumination when employing the holding focus. It
is not compatible with Temp inserts and CO2-incubators.