Zeiss LSM 880 with AiryScan FAST Guide de configuration
Training Guide for Carl Zeiss LSM 880 with AiryScan FAST
ZEN 2.3
Optical Imaging & Vital Microscopy Core
Baylor College of Medicine (2018)
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2 – LSM 880 Training Guide
Power ON Routine
1
Turn ON ‘Main Switch’ from the
remote control paddle.
2
Turn ON both ‘Systems/PC’ and
‘Components’ switches.
By default, the ‘Lasers’ key located
on the side of the remote paddle
should always be in the ‘ON’ state,
but please verify.
3
Turn ON HXP-120V epi-fluorescence
light source.
This light source is required for
visualizing fluorescence via the
microscope eyepieces.
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3 – LSM 880 Training Guide
Power ON Routine
4
Turn ON HP Z840 PC.
Wait for PC to boot into Windows
OS and login to ‘oivm’ user account.
5
Double click ZEN Black icon to start
software.
Note: Be sure to use Zen Black
instead of ZEN Blue. Icon above is
for Black version.
6
When software has started, you will
be presented with a login screen.
Select ‘Start System’ to scan new
images. If processing existing
images, select ‘Image Processing’.
Wait for initialization of system to
complete.
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4 – LSM 880 Training Guide
Getting Started with ZEN 2.3
ZEN 2.3 is separated into three distinct areas.
Image Acquisition Tools
Represented by a group of blue bars each containing a series of tools for sample observation, image acquisition, image
processing and system maintenance.
Image Display
This area is where each newly captured image will appear. Settings here allow the user to control how the image is viewed
on screen.
Catalog of Open Images
This list displays each image that is currently open within the ZEN software. This area contains tools for saving data sets.
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5 – LSM 880 Training Guide
Getting Started with ZEN 2.3
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6 – LSM 880 Training Guide
Powering ON Lasers
Before we begin setting up the software for imaging, we first need to verify that our lasers are powered on. There is a
considerable wait time for the Argon laser to warm up (5 minutes) so please complete these steps first if the system is being
powered on from an off state.
1. Select ‘Acquisition’ from the main toolbar.
2. Expand the blue toolbar labeled ‘Laser’ and select ‘Argon’ from the laser list.
3. Toggle ‘Power’ button from Off to On.
Note: This laser requires a 5-minute warm up time. Once you select
‘On’ the system will count down from 300 seconds. You will not be able
to use the laser until this warmup is complete.
4. Turn ON remaining lasers (as required) from the software dialog
‘Laser’.
Note: The 405 laser is direct modulated and automatically powered on
when the laser is selected in the light path dialog.
The 561, 594 and 633 lasers can be powered on by selecting each laser
and switching from Off to On state.
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7 – LSM 880 Training Guide
Mounting Sample on the Microscope
While the lasers are warming up, we can mount our specimen on the stage above the microscope objective.
1. Place sample on microscope stage above objective (roughly in the
center of the field of view).
2. Select the ‘Locate’ tab in the Image Acquisition Tools.
3. Use the Shortcut buttons to select filter set for visualization in the
eyepieces.
Green
– generic green filter set
Red
– generic red filter set
Blue
– generic blue filter set
TL/DIC
– transmitted light
All Closed
– turn off all light sources.
Note: These shortcut buttons will automatically select the filter set and
open the light source shutter.
Alternatively, you can turn on the epi-fluorescence light path manually
using the ‘Microscope Control’ tab located below the configuration
shortcuts. Here you can turn on/off the epi-fluorescence light source (A),
change your filter set (B) and control transmitted light (C) if applicable.
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8 – LSM 880 Training Guide
Mounting Sample on the Microscope
4. Via the microscope eyepieces, visualize your sample and adjust the focus (Z) and stage position (X,Y) accordingly.
5. Once a suitable location is found, click ‘ALL CLOSED’ from shortcuts (3).
6. Select ‘Acquisition’ from main toolbar to begin imaging.
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9 – LSM 880 Training Guide
Light Path Configuration
The confocal portion of the LSM 880 is equipped with a 34-channel spectral detector plus transmitted light and has 5 lasers with
wavelengths 405nm, 458nm, 488nm, 514nm, 561nm, 594nm, and 633nm. The Light Path configuration allows you to program
precisely how your image acquisition will be executed.
The first step is to decide how you wish to collect each fluorophore. You have 3 choices:
1.
Simultaneous
– collect up to 4 fluorophores simultaneously.
Note: While this mode is the fastest approach – many fluorophore combinations will exhibit enough spectral overlap to be
able to detect unwanted signal from neighboring fluorophores using this method. This phenomenon is known as spectral
overlap or ‘bleed’.
2.
Sequential
– collect fluorophores sequentially.
Note: This mode tends to be the slowest mode – but provides accurate spectral separation of multiple fluorophores.
3.
Spectral
– lambda mode allows you to collect fluorophores simultaneously and unmix based on reference spectra.
Note: This mode is both the fastest AND the most accurate mode of spectral separation, however it requires single label
controls to differentiate between the unique spectral trace(s) from your fluorophore(s).
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10 – LSM 880 Training Guide
Light Path Configuration
In the following example, we will design a sequential acquisition of two fluorophores (Alexa 568 and Alexa 488).
Zeiss uses ‘Tracks’ to group a series of settings specific to a single fluorophore. Using sequential imaging, you should have one
track for each fluorophore. These tracks can be switched in one of two ways:
1.
Line
– track settings are switched after scanning each X line in the image.
Note: Line track switches minimize the time lag between colors, however no hardware settings are permitted to change
between tracks apart from laser line and detector choice. All other settings must remain the same. This limits you to a
maximum of 4 fluorescence channels plus one transmission image with this mode.
2.
Frame
– track settings are switched after scanning the entire XY frame for each image.
Note: Frame switch introduces the most time lag between colors, but the user can change any hardware setting between
tracks, even using the same detector with different settings. You must use Frame for more than 4 color imaging
sequentially. Also note that Zeiss introduces a 750ms delay between each track change to allow for mechanical changes.
You must add this to the acquisition time between each color.
Decide on which track mode (line or frame) you will use prior to setting up your light path.
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