Zeiss LSM 980 Airyscan 2 - Part 2.2: How to acquire an image in "Airyscan" mode .

Make sure the "Auto Save Path" is set to the local Data (D:) drive.

In the "ZEN" software go to Tools/Options.

Choose "Saving" and check the "Path" and adapt accordingly if needed.

In order to see all needed options in the "ZEN" software always activate "Show All (Global)" under "View".

These settings are saved to your profile and should persist even after log-out or restarting the microscope.

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

Identify the following sections in the "Acquisition" panel:

Control panel including beam path and acquisition parameters.

Image display.

Display adjustments.

Gallery of acquired images.

Hardware controls as objectives, lasers, stage e.g. .

Click "Smart Setup" to open the "Smart Setup" wizard.

Click "+" to add dyes/channels to your experiment.

Select the detector for "Airyscan" imaging.

In this tutorial we only cover the "Airyscan" acquisition. For "LSM" acquisition please refer to the corresponding guide.

Choose a Scan Mode:

Airyscan SR/CO: max gain in resolution and/or SNR, pinhole default 0.2 AU. Full Airy Detector will be used (total detection area 1.25 AU).

Airyscan Multiplexing (MPLX) SR-4Y/SR-8Y/CO-8Y: increased resolution and/or SNR and increased speed. Here beamshaping along the y-dimension to allow for 4x or 8x parallelization (4 or 8 image pixels are scanned in parellel).

For detailed information on the different modes please have a look here: Zeiss-Microscopy Technical Note.

Finally evaluate the speed/signal tradeoffs and select the optimal experimental imaging strategy. Usually the "Best Signal" (Frame sequential) is a good choice for starting.

Click "OK" if satisfied.

The software automatically sets all the needed tracks and corresponding wavelength settings. Lasers are getting switched on automatically.

In the menu box "Imaging Setup" one can review and adjust/optimize the chosen imaging setup if necessary.

One can review/adjust the Emission filters (Dual band-pass) for each track and/or assign alternative filters.

Try to fit he filters omitting as good as possible any bleed-through between channels.

Further, you can specify the sequential strategy "Switch track every -" ("Line", "Frame fast" or "Frame") and adjust MBS (main dichroic beam splitters) if needed.

"Line" and "Frame fast" sequential scanning mode require same hardware between all tracks for maximal speed. At the emission filter wheel "Plate" has to be selected.

"Frame" sequential scanning allows changing hardware (Emission filters, MBS e.g.) between tracks/frames.

In the "Channels" box you can check which laser line has been chosen for each "Track".

In the "Channels" box you can adjust the laser power, the diameter of the confocal pinhole as well as the detector master gain.

Optimal performance of the detectors is achieved when using a master gain of 650-900V.

If working in MPLX mode (vs SR mode) the equivalent laser power in confocal mode is shown here (due to the beam shaping in MPLX mode).

In the "Acquisition Mode" box you can change the scan area, adjust the scan speed as well as optimize the image size for optimal sampling and therefore image resolution. They might differ between SR or MPLX mode. See steps ....

Select a "Track" for adjusting in the "Channels" box by just clicking on it. It becomes highlighted.

Click "Live" for starting the acquisition.

Adjust the "Display" histogram by clicking "Best Fit" for seeing an image.

Refocus by using the hardware focus wheels or by holding "Ctrl" and using the mouse wheel.

Optimize the image quality by adjusting the detector gain as well as the laser power.

Adjust laser power (3) and Master Gain (4) (700 – 900) (5) Shows the equivalent laser power compared to confocal mode.

Usage of full dynamic range not necessarily needed with Airyscan. (image missing)

Check the histogram (peak at the highest grey level) and/or tick the "Range Indicator" (red pixels) to check for saturation.

You can adjust position while live scanning within the software:

Simply double-click on the desired area in the live image and this particular positions gets centered.

Click at the outer edges moves the stage in that direction.

Depending on thr Airy mode choosen different scan parameters can be set.

Open the "Acquisition Mode" box.

Change your field of view by using either the zoom factor (the area of interest should be centered) or choosing "Crop Area".

The minimum zoom factor/scan area should be 1.7x.

Place, rotate and adapt the crop area according to your needs.

"Frame Size" defines the number of pixels in one scan area.

Click "Confocal" to optimize pixel size according Nyquist criteria sampling (gets highlighted if active). Proper setting of the xy sampling (pixel size) is crucial for acquiring optimal images.

Increases acquisition time if large field of view.

Change the "Scan Speed" by adjusting the slider or clicking "Max" (always uses fastest possible scan speed independent of changing zoom or frame size) (highlighted if active).

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

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

Please make sure it is properly aligned. Otherwise press the "Auto" button.

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

"Mean Intensity" ("Repeat per Line or per Frame"): may be used to remove noise (if high gain e.g. ...)

"Sum Intensity" ("Repeat per Line or per Frame"): useful for very weak signals.

If applied acquisition time will increase.

Select another "Track" in the "Channels" box to adjust those settings. Repeat for all available "Tracks".

You can switch between "Tracks" while being "Live".

Finally press "Stop" and click "Continuous" for a live view of all channels as well as using the resolution as defined in the "Acquisition Mode" box.

If adjustment is necessary, untick the other tracks for ease of use and faster scanning.

Once you are satisfied with your current settings for each "Track" click "Snap" to acquire an image.

Right click on the file in the "Images and Documents" box.

Save the image on your ZMB data folder.

Activate the "Z-Stack" checkmark.

Press "Live" and focus through your sample to set/adjust the z-stack boundaries ("Set First" and "Set Last") in the "Z-Stack" menu box.

Click "Optimal" in the "Z-Stack" menu box for automatically optimizing the z sampling ("Interval" = z-step size).

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

Click "Start Experiment" for acquiring the defined "Z-stack".

After acquisition you can navigate trough the slices of your stack in the "Dimensions" tab.

Save your stack the same way as you saved the image in the last step.