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
IV 000000-2071-464 10/2014 V_01
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
10/2014 V_01 000000-2071-464 1-I
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
1-II 000000-2071-464 10/2014 V_01
•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
LSM 880 Contents ZEISS
10/2014 V_01 000000-2071-464 1
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

CHAPTER 1 - SYSTEM OPERATION
ZEISS Contents LSM 880
2 000000-2071-464 10/2014 V_01
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

CHAPTER 1 - SYSTEM OPERATION
LSM 880 Contents ZEISS
10/2014 V_01 000000-2071-464 3
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

CHAPTER 1 - SYSTEM OPERATION
ZEISS Contents LSM 880
4 000000-2071-464 10/2014 V_01
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