LAS X
for Confocal Systems

Release Notes LAS X 4.1.1

Leica Microsystems CMS GmbHAm Friedensplatz 3
D-68165 Mannheim, Germany

Release documentation for LAS X 4.1.1
This document describes the 4.1.1 release of the Leica Application Suite X imaging
and analysis software for advanced live cell research. You should read this document
before installing a copy of this software.
All reasonable steps have been taken to ensure that this publication is correct andcomplete. Should any user be in doubt about any detail, clarification may be soughtfrom Leica Microsystems CMS GmbH, or their accredited representatives. The
information in this document is subject to change without notice and should not be
construed, as a commitment by Leica Microsystems CMS GmbH. Leica Microsystems
CMS GmbH accepts no responsibility for any errors that may appear in this document.

Copyright © 2020 Leica Microsystems CMS GmbHAll rights reserved. The contents of this publication may not be reproduced in any
form, or communicated to a third party without prior written permission of Leica
Microsystems CMS GmbH. Due to a policy of continuous development; we reserve
the right to change specifications without notice. Microsoft and MS-DOS are registeredtrademarks and Windows, the Windows logo, the Windows 2000 logo, the Windows
XP logo, the Windows Vista logo, the Windows 7, and the Windows 10 logo aretrademarks of Microsoft Corporation.
Date: October 23rd, 2020 applying to build number 23273

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Contents
1. Compatible Microscopes and Technical Requirements ............................................................................ 4
1.1 Compatible Microscopes and Hardware Requirements .................................................................. 4
1.2 Operating System Requirements ........................................................................................................ 4
2. Reason for this Release – LAS X 4.1.1 ........................................................................................................ 4
3. New functionality and features ...................................................................................................................... 5
3.1 Support of the DLS modality ................................................................................................................ 5
3.2 Support of the STED modality ............................................................................................................. 7
3.3 Miscellaneous novel features .............................................................................................................. 9
4. Recommendations ........................................................................................................................................ 10
5. Open Issues / Restrictions ........................................................................................................................... 10
6. Solved Issues / Restrictions ......................................................................................................................... 14
7. Compatible 3rd party software ...................................................................................................................... 14

LAS X 4.1.1 Release Notes for Confocal Systems

31. Compatible Microscopes and Technical Requirements
1.1 Compatible Microscopes and Hardware Requirements
The LAS X 4.1.1 control software is a release for the STELLARIS confocal platform.
Accordingly, the LAS X 4.1.1 control software is not compatible with any othermicroscope platform from Leica, i.e., SP8 confocal systems also cannot be upgradedfrom LAS X 3.5.7 with this release. Note that LAS X 4.1.1 supports DLS, FALCON and
STED, but not any other advanced imaging modality of the STELLARIS platform(CARS, DIVE, FCS). All Leica-supplied CUDA workstations for the STELLARIS
confocal platform meet the minimum PC requirements of the LAS X 4.1.1 control
software.

1.2 Operating System Requirements
LAS X 4.1.1 is a genuine 64-bit program and runs on Windows 10 64bit. Since the endof the lifetime of Windows 7 in 2020, LAS X 4.1.1 does not support Windows 7
anymore.

2. Reason for this Release – LAS X 4.1.1
LAS X 4.1.1 is a release to address vulnerabilities in a 3rd party application (WIBU
CodeMeter Runtime) shipped with LAS X for license management.

LAS X 4.1.1 Release Notes for Confocal Systems

43. New functionality and features
In this Release Notes (LAS X 4.1.1), the following section about New functionality
and features is identical to that of the previous Release Notes (LAS X 4.1.0). It
informs the user about the new functionality coming with LAS X 4.1.x.

3.1 Support of the DLS modality
The LAS X 4.1.1 control software supports the DLS (Digitial LightSheet) module toextend the STELLARIS platform with a gentle and fast lightsheet imaging modality.
DLS can be operated via an intuitive wizard (see figure LightSheet wizard –LightSheet tab).

LightSheet wizard – LightSheet tab
Two-color recording of a zebrafish embryo with DLS in the xymzt mode. The red box on the lower left indicates the Widefieldbutton for switching quickly to the Widefield acquisition mode; the red box on the top indicates the Widefield tab.

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With STELLARIS, we introduce several novel functionalities for DLS:We added a new Widefield mode for quickly navigating through thespecimen. The Widefield mode can be used within the LightSheet tab
or in the designated Widefield tab of the LightSheet wizard.

There is now the possibility to generate the DLS lightsheet with theresonant scanner. Due to shorter pixel dwell times, using the resonant
scanner will result in even more gentle imaging paradigms. Users canoperatethegalvo/resonant

scannerswitchinCalibration>

Optimization > Acquisition. It is possible to change the scan mode
within the current LAS X session.


We added an illumination wavelength-specific calibration for thelightsheet to correct for chromatic aberrations. Users can do the basecalibration in the Calibration tab and the channel-specific calibrationin the LightSheet tab.



Since data volumes can quickly grow large in DLS experiments, wewould like to point out the novel Compatibility mode for large
experiments for recordings of large data sets. This data mode allows
for saving experiments larger than the caching folder (DataContainer).

We fully integrated the ImageCompass into the Confocal tab of theLightSheet wizard, i.e., users can set up (high-res) confocal scans justlike in the main STELLARIS interface.

A comprehensive description of how to operate the DLS wizard (including the abovementioned features) can be found in the Online-Help.

LAS X 4.1.1 Release Notes for Confocal Systems

63.2 Support of the STED modality
The LAS X 4.1.1 control software enables STED nanoscopy on STELLARIS STEDand STELLARIS 8 STED. The STED modality is seamlessly integrated into the
ImageCompass user interface: Users can intuitively define even complex imaging
paradigms via Drag&Drop (see figure ImageCompass STED – confocal/STED
comparison and ImageCompass STED – Drag&Drop).

ImageCompass STED – confocal/STEDThe STED modality is seamlessly integrated into the ImageCompass: The unified interface allows for setting up
confocal/STED comparisons (upper and lower setting) as well as multi-color STED experiments (not shown) with ease.

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ImageCompass STED – Drag&Drop
When choosing a STED-able dye, users can choose between the confocal (C) and STED (S) modality by clicking on therespective small flag (a), and subsequently Drag&Drop the dye (b) into a setting. The ImageCompass will configure thehardware automatically for STED dyes (i.e., excitation wavelength from the white light laser (c), STED laser line (d), andemission range of the detector), so that the user only needs to fine-tune some Beam Path settings (such as the laser powerof the white light laser and STED laser, or the STED 3D parameter (e)). STED is compatible with all members from the PowerHyD family (here, a HyD S detector is used).

STED supports our novel TauSense technology: Simply choose a mode from the
TauModesdrop-down

menu

(see

also

figureImageCompass

STED

–

confocal/STED for the TauModes menu in the upper setting). The various TauSense
modes allow for powerful, digital gating to increase STED performance (GateScan,
TauGating) and exploit life-time based information (TauContrast, TauScan,
TauSeparation).
Furthermore, on STELLARIS 8 STED FALCON, -STED is available.-STED uses
lifetime information in combination with phasor analysis to increase the resolution
and eliminate uncorrelated background in an automatic manner. Since much lessexcitation and STED laser power are necessary, -STED can even enable gentle
live-cell imaging with cutting-edge resolution and high image quality.
A comprehensive description of how to operate the STED modality (including Autoand Expert Beam Alignment, TauSense and -STED) can be found in the OnlineHelp.
LAS X 4.1.1 Release Notes for Confocal Systems

83.3 Miscellaneous novel features
FRET wizards
The FRET AB (Acceptor Bleaching) and the FRET SE (Sensitized Emission) wizards
are now available with the MicroLab license. The first step of both wizards (Overview)provides a guideline on how to perform the respective FRET workflow: From Setup(using the ImageCompass) to Evaluation (generating insights about the FRET
efficiency).

FLIM zip
For STELLARIS 8 FALCON, a new FLIM data format is now available: FLIM zip.
FLIM zip works both online and offline. It optimizes the file size for faster analysis
performance and improved data storage (up to 8 times smaller file size). FLIM zip
can be activated with a right-click on an acquired image (once activated all theimages will be acquired as FLIM zip) or in the Tool menu. With the latter option, it ispossible to optimize already acquired FALCON data with FLIM zip (i.e., dataacquired on LAS X 4.0.2 or lower), however, FLIM zipped data can only be handledwith LAS X 4.1.0 or higher.

TauSense improvements
TauSense describes several acquisition tools which use pulsed laser excitation andphoton counting detectors to obtain fluorescence lifetime-based information for image
formation. With LAS X 4.1.1, TauSense modes are further integrated into LAS X. Now,
stage applications (such as the Navigator) can use TauScan and TauSeparation.
Furthermore, we have refined the metadata handling of the TauSense technology(improved channel labeling and image properties).

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4. Recommendations
For an optimal performance and the safety of the product, we strongly recommend theinstallation of/upgrade to LAS X 4.1.1 for systems of the STELLARIS confocal
platform!

5. Open Issues / Restrictions
The following list enumerates the known, unresolved anomalies of LAS X 4.1.1:

Bleach point not available in the core software

Workaround: The bleach point functionality allows for applying a high laser
intensity to small defined regions within the specimen so that FRAP or photoconversion experiments can be performed. Since the bleach point functionality inthe core software will only be implemented in an upcoming LAS X release, for thetime being, there are two mitigation strategies:
o If the temporal domain is not critical (e.g., photo-conversion of fluorescentproteins for long-term cell tracking), a simple solution is using small ROIs
to manually zoom-in and apply high laser intensity.
o The FRAP wizard contains the bleach point functionality. For more
complex experimental conditions in which the temporal domain matters,
get in touch with us so that we can assist you with a license for MicroLab.



No frame accumulation with TauContrast imaging

Workaround: Enabling frame accumulation results in images with an improved
signal-to-noise ratio (SNR). Since frame accumulation will only become available
for TauContrast imaging with an upcoming LAS X release, an immediate
mitigation strategy is the use of line accumulation in TauContrast imaging.

LAS X 4.1.1 Release Notes for Confocal Systems

10Indeed, line accumulation even has the advantage of being better suited for fast
imaging since it minimizes the interval between the iterative recording of thesame pixel. However, consider that line accumulation will allow for less relaxationof fluorescent dyes/proteins between repetitive excitation cycles, and thus. a
more cautious approach regarding the photon budget should be considered.



No Dynamic Signal Enhancement with TauSeparation

Workaround: Dynamic Signal Enhancement (DSE) is an image filter for noise
reduction in time series experiments. It is particularly useful for fast life-cell imaging
with the resonant scanner, as it reduces noise without sacrificing much of thetemporal resolution. Since DSE will only become available for TauSeparation
imaging with an upcoming LAS X release, line accumulation can be used fornoise reduction as a temporary mitigation strategy. Since line accumulation does
decrease the temporal resolution (proportional to the number of times a line isaccumulated), several measures and/or a combination thereof should beconsidered for minimizing the acquisition time of individual image frames. These
measures include: Minimize line accumulation by “hardware means” (high-NA
objective,

appropriate

detector,higher excitation

power),

minimizeline

accumulation by “lower resolution” (larger pinhole, smaller image format), use of
the resonant scanner (shorter pixel dwell times), and a smaller field of view (cropalong y- and/or x-axis).



Unused disk memory blocks new experimentWorkaround: If unused disk space is not freed after an experiment is executed,
the user should close and reopen the project.



LAS X freezes/unresponsive

Workaround: If software performance slows down or even freezes due to highand prolonged workload, the user should restart the LAS X (software) andscanner (hardware). Closing of the LAS X session may require using the Windows
Task Manager.
LAS X 4.1.1 Release Notes for Confocal Systems

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Frequency of the White Light Laser (WLL) wrongly displayed

Workaround: The WLL frequency in the GUI is not synchronized correctly withthe hardware control on LAS X start. Therefore, the user must once change
the frequency of the WLL to correctly sync the GUI and the hardware control.
This will solve the issue for the remaining LAS X session.

Optimize format not working correctly for STED

Workaround: The format optimization does not work correctly for STED and thus
the user should set the format (pixel size) manually.



Selection of different detector modes in STELLARIS 8 FALCON for HyD S
possible

When FLIM is started, the appropriate detector mode (‘counting’) is
automatically activated on HyD S. Nevertheless, users are not prevented from
actively choosing an inappropriate detector mode (‘analog’ or ‘reflection’) on HyD
S while FLIM is on.



Average Arrival Time (AAT) changed when LIGHTNING applied to an image
with TauSense

Workaround: Since AAT data is changed in LIGHTNING, the user must use theraw data (i.e., not deconvolved) for analysis of the AAT channel.

Saving with .xlef/.lof in FALCON

Workaround: Due to the newly introduced FLIM zip, FALCON data can currently
only be saved reliably as LIF file. LIF is now the default format for saving in LASX.

LAS X 4.1.1 Release Notes for Confocal Systems

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FRET efficiency image does not match the statistic

Calculation of the FRET efficiency statistics works correctly while the FRET
efficiency image may not be displayed correctly and thus should not be used.



Only single histogram is generated when using TauSeparation in combination
with multiple settings and line sequential

Workaround: When using TauSeparation with multiple settings and line
sequential, only a single (identical) histogram is generated for all settings.
Nevertheless, the generated image data is correct and can be used for further
analysis. If the histogram data is important, the user must choose frame
sequential mode for TauSeparation with multiple settings.



Galvo switch (from resonant to FOV scanner) should not be used in the
LIGHTNING wizard

Workaround: When using the Galvo switch (from resonant to FOV scanner) in the
LIGHTNING wizard, the software creates an error. Accordingly, the user must not
operate the Galvo switch inside the LIGHTNING wizard. If the user accidently
operated the Galvo switch, the user must close the LIGHTNING wizard and set
up reasonable scan parameters (Speed) for the FOV scanner in the main
STELLARIS interface before returning to the LIGTNING wizard.



FRAP wizard: “Overview” tab button malfunction

Workaround: “Overview” tab in FRAP wizard must not be clicked since it
causes a scan loop. The information from the “Overview” tab can be found in theOnline-Help. If the “Overview” tab is accidently clicked, the user can terminate theinitiated scan by clicking on the ”Close X” at the top right of the LAS X and thenreturning to the Setup tab.

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6. Solved Issues / Restrictions
Amongst other bugfixes, we have resolved the following issues/restrictions from theLAS X 4.0.2 release:

FRET wizards not available



TauSeparation/TauScan not compatible with Navigator and TileScan

TauScan values not fully visible in image propertiesGUI elements not consistently explained via Mouse over

LDM lacks Trigger functionality

Save progress bar does not disappearData Export of TauContrast images does not work (e.g. as .tiff)

STELLARIS systems without Tandem Scanner have Galvo switch icon in GUI

7. Compatible 3rd party software
LAS X 4.1.1 does not support the integration of any 3rd party software packages, i.e.,neither SVI’s Huygens Suite nor PicoQuant’s SymPhoTime. The functionality of thesepackages is covered by the LAS X LIGHTNING and LAS X FLIM packages.

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