STARTUP AND SHUTDOWN INSTRUCTIONS FOR LEICA STELLARIS
as of 2022-07-12

A few rules:

• Sign up times are strict.
• All files must be stored on Drive E:  Files stored anywhere else will be considered evidence that the system overall is not being used properly; ref Van Halen and brown M&Ms.
• Lenses have different immersion media (air, water, gycerol, oil). Make sure you use the correct one.
• All immersion media bottles must be placed in secondary containment. Keep bottles clean. Do not drip. Do not place bottle on any surface other than the secondary containment. Messy oil bottles is indicative of messes eleswhere.

Simple startup:

Three power switches.
The order is not important, but the computer and microscope hardware must be communiating before any software is run.
Do not put sample on microscope until everything is turned on.

• Power strip strapped to pillar behind monitor on right is power for computer and accessories. The computer will boot automatically when this switch is turned on. You can tell when the computer is on because the labels about the bar of knobs on the table will display text.
  Troubleshooting: Maybe the person before you did not turn this off. Throw the switch a second time.
• Main control box on floor, at left top, switch marked Power.
• Main control box on floor, at left top, switch marked Laser.
• Additional power for environmental chamber and other optional accessories will be added later.
• If both monitors do not turn on, go to bottom of this web page to Troubleshooting section.
• Log in to computer as LAS-USER [need to check this] with password posted on the moitor or look under the keyboard.
• LASX software.
• A small window will pop up asking for configuration (the window may pop up on a different screen, so look for it).
  "Confocal and MP" is the standard. "Confocal" also works without the MaiTai laser.
• Load settings (down arrow button), reuse settings from an image (right click and apply), or set up hardware manually.
  To set up manually, turn on lasers and set spectral detections. As of writing this, we recommend fully manual application of these settings, but Leica does have menus of presets.
• Optional: load or assign your favorite knob settings.
• Lens may be selected from LASX or from touchpad on front of microscope body. When lens button blinks, this is a reminder that you are switching to a different immersion lens; press a second time to engage.
• Before putting sample on microscope, using focus knob on microscope go to low position. Follow these instructions.
• Touch pad on front of microscope controls lighting mode for looking by eye.
• If you are using confocal to collect transmitted light image or second harmonics with the transmission detector, then you must align the condenser for Kohler illumination and open the field diaphragm fully.

There are two imaging modes on this system. The one more like other confocals or a camera system is when the detectors are set for "intensity" or "digital." There is also a photon counting mode.

In both modes, you do not need to fill the entire dynamic range. You may use a portion of the dynamic range and boost the contrast. If you choose to use the full range, DO NOT SATURATE the image.
For visualization only, to see dim and bright scaled together, there is a gamma LUT available in the histogram window at the lower left of the image window.

If you have multiple conditions where intensity relationships are important or you are checking controls, you must start by setting intensity range for the brightest condition and leaving it there for all conditions. DO NOT SATURATE.

We have been having occasional bug that the smart grain &/or smart instensity knob stops working, there is a message it is disabled.
First check that the correct image pane is active and that the laser and detector are both on in the correct mode.
If these do not solve the problem, then delete the track and reload the setting.
Laser intensity may also be set by clicking on the line in the track which will open a small settings window. Detector gain may be set similarly.

When taking tiled or mosaic images, beware the Task window in the lower right of the screen. This is your friend when you want to collect multiple regions automatically, but it is you enemy if you have multiple regions and you only want to collect one at a time. Before capturing the tiled region, be sure to only have the tasks you want executed checked. (And see below "Saving files")

Saving files

Treat each project as a container for each sample or condition.

Name once. Files inside do not need to be named or could be called "Z series"

Each time switch samples, start a new project.

However, these files can get large, many GB. So may want to make a new project for the same sample to keep the file sizes low.

Also, issues of subdirectories within LIF files. These occur with tiling and FLIM. These do not cooperate well with importing into ImageJ, Imaris, etc.

Therefore, for tiled files, copy/paste or cut/move only the "merged" files to another project.

If you are comfortable that you only need the merged tiles file, you could delete the individual images from the project and only keep the merged files. (This will also halve the data size.)

For FLIM, we haven't worked out the details, but each run may be a different project.

Shutdown

If somebody is coming in immediately after you, you may leave the entire system on, including the lasers they will be using.
If they will not be using all the lasers, turn off the ones they will not be using.

TURN OFF LASERS MANUALLY BEFORE QUITTING SOFTWARE.

If you forget, restart the system and turn off the lasers before final shutdown.

Bring focus down before turning off microscope.

Troubleshooting

If both moniotrs do not turn on, manually turn on with the power switch in back.

Buttons on rear of display monitor. Vertical rectangle button at bottom is power. Turning this on should connect the monitor to the computer and automatically be recognized as a second screen. Four smaller square buttons are used for maneuvering through the sesstings menus.

Monitors can be reporgrammed to turn on when power starts up:
1. Turn on the monitor.
2. Use monitor buttons positioned at the right back and make sure that the following settings are set: • Main Menu → Management → DDC/CI Support: OFF • Main Menu → Input Control → DP Hot-Plug Detection: Always Active
3. Turn the monitor off and on again. This ensures that the setting modifications are applied.

other notes...

LAX automatically initializes stage on startup, so no sample on the scope!
Top left of screen, Configuration, Turn on laser.

Gear Hardware, Bit Depth 16.
Highlight detector in slider to turn on /off in lower right
Add Laser, turn shutter on in tool with lines and bullets.
For MP and harmonics tracks, pinhole wide open.
For reflection imaging, look in AOBS settings.