Leica Stellaris 5 - 2: Acquire an image in xy and z

Go to "Configuration".

Select "Laser Config".

Switch "ON" the lasers you need.

The WLL is set to 85% by default and allows selection of excitation wavelengths from 485 nm to 685 nm.

Go to "Hardware".

Here you can change the image "Bit Depth" if necessary.

Go back to "Acquire".

Make sure your sample is correctly placed and focused. Check the appropriate Start-up guide for more info.

Identify the following sections in the "Acquire" panel:

Make sure you are in the "xyz" scan mode.

Settings for "XY" ("Format", "Speed" e.g. adjustments).

Settings for z ("Z-stack" option).

Excitation settings.

Objectives.

Detectors and emission settings.

Type to search for your fluorophore/dye of interest.

Available results will be displayed here.

Drag the suitable one into the empty panel ("Setting 1").

The corresponding laser line will be automatically added.

Repeat the same procedure for the remaining fluorophores by adding them to the same panel ("Setting 1").

Click the "Open Dye Assistant" button.

"HyD S" Detectors are used for regular acquisition.

Choose the most appropriate configuration from the different options - avoid having crosstalk!

"Line sequential" acquisition is faster compared to "Frame sequential".

For TauSense acquisition see the appropriate guide.

Click on the different detectors to adjust imaging parameters.

Set detectors to "Counting" "Operating Mode".

Here you can also select your dye to visualize the emission spectra.

Toggle to activate/deactivate the excitation spectra as well.

Double click here to change look-up table (LUT) for each active detector.

You can use the "Fast Live" button to quickly adjust focus or zoom.

Only images from the active sequence will be shown while live.

Click the clock wheel to adjust "Fast Live" settings (each scanner has independent settings).

Resonant scanner is NOT available at Stellaris 5 inverse (USZ).

Adjust laser intensity by double clicking and typing in.

Hovering over the laser bar and rolling the mouse wheel also allows intensity adjustment.

These settings are only used to allow a fast screening / localization of your sample. You will need to readjust proper imaging setting under "Live" view for image acquisition.

Start adjusting your image sequences/settings.

Click "Live".

Adjust laser power.

Adjust the image contrast by using the slider or activate automatic contrast adjustment ("Auto Contrast").

Click here to reset the contrast adjustment to full scale.

Activate the image histogram to to inspect the intensity distribution and to optimize your signal. Avoid under- and overexposure.

For counting mode adjust to approx. 80 counts / usec pixel dwell time (keep an eye on the pixel dwell time - depending on the set format and scan speed).

Proper setting of the xy sampling (pixel size) is crucial for acquiring optimal images.

Change your field of view by using the "Zoom". You can also use the knob in the external control panel.

"Format" defines the number of pixels in one scan area.

One can choose preset formats via the drop down menu.

By clicking the "+" every other format can be chosen.

To adjust for the correct pixel size/optimal sampling you can either use the online calculator such as the SVI Nyquist Calculator or the auto button for an estimate.

Here you can change the scanning speed:

via the drop down menu (presets) or via "+" for every other scan speed.

Use slower scan speed to increase the pixel dwell time and thus collect more light.

Increased scan speed leads to faster imaging, lower photo-damage and bleaching, but gives more noise and allows a smaller field of view.

Toggle between the resonant and galvo scanner here. It might take a few seconds to adjust after switching.

This option (resonant scanner) is NOT available at Stellaris 5 inverse (USZ).

You can also activate "Bi-directional" scanning to speed up acquisition.

Please make sure the phase is properly adjusted.

If you are limited by the laser power but still need to increase the signal (or reduce noise) use:

Accumulation (by line or by frame): Useful when using detectors in counting mode or for very weak signals.

Averaging (by line or by frame): may be used to remove noise.

If applied the acquisition time will increase.

Use the z-drive controller ("Z-Position" on the "control panel" to define the limits with "Begin" and the "End" of your z-stack.

Define the appropriate "Z-Step size" or go for optimal z sampling by choosing "System Optimized". The "Number of Steps" will be automatically calculated.

You should refer to the SVI Nyquist Calculator if you plan to deconvolve your image as a post-processing step.

Press "Start" to begin acquisition.

You can adjust contrast by using the slider.

Go through the different z planes here.

Use the orthogonal viewer to inspect your z stack.

You can also use the 3D viewer here.

Go to the "Open projects" tab.

Click on your data file and give it a meaningful name.

Click on the disk button to save the data. Alternatively right click and choose "Save as..." from the drop down menu.

Save the data to your zmb-data network folder.