LSM 880 Airyscan Module

Microscope differences between standard confocal and Airyscan imaging

Using Airyscan (activating "ChA" at the top-right of the beam path) disables the use of other detectors.

The Airyscan detector array cannot select an arbitrary detection window, but uses filter cubes (the position just before the Airyscan detector in the beam path)

The pinhole setting becomes irrelevant, as Airyscan uses its own, default pinhole setting - which is not displayed on the "pinhole" slider in the software.

Available modes:

• SR uses all detectors
• Res/Sens hands in some resolution to achieve a higher signal-to-noise-ratio (SNR)
• Virtual Pinhole allows simulation of different pinhole sizes (eg. to allow picking up weaker signals by expanding the PSF)
• Confocal allows for normal confocal imaging, using only the central detector (like in a normal confocal)

The system requires calibration

• Airyscan needs to be calibrated
• To do this, find a region in the sample with high signal intensity and broad distribution throughout the field-of-view (FOV)
• If weak signal exists, boost laser power and Airyscan detector gain, to achieve higher intensities
• Use "continuous" mode for a while, and in the image preview area of the software, click on the "Airyscan" tab to show the distribution of intensity across the detector
• Let the software auto-calibrate, until the central detector is the brightest - then move on to the actual experiment.

Processing is necessary to achieve the final high resolution image

• Airyscan images need to be processed, before coming to the final image that has the increased resolution and SNR.
• In the global "Processing" tab of the software, find and select "Airyscan processing"
• Select your image in the library on the right, and click "select" on the left
• A good starting reassignment value is "Auto"; but when wanting to compare intensity values between images, it would be good to take the "Auto" generated value from one image, and manually apply it to all to-be-compared images.

Fast Airyscan is fast, but with less resolution

• Fast Airyscan is slightly different, as it uses the central column of detectors, and splits the excitation laser into a vertical row of 4 lasers, which scan the sample in a paint brush-like manner.
• It loses some resolution, but gains a lot of speed. The resolution and SNR benefits, still outperform standard confocal.
• Enable Fast mode on the left, above the beam path and detector windows.
• Adjust laser power to ca. 4x the normal, expected required laser power, as it will be distributed across 4 beams.
• Calibrate the system in this mode, again by using "continuous" mode on a high signal region, and monitor the calibration in the "Airyscan" tab of the image preview display

General notes on using Airyscan

• As Airyscan genrally requires higher laser intensities, it is not a gentle imaging mode, but still suitable for live-cell imaging if properly adjusted for the experiment.
• Note that for all channels, you need to separately optimize Airyscan, and note that frame-wise, multicolor sequential scanning is only possible when using one filter cube for both channels (eg. which allows GFP and RFP light through).
• Very useful for Airyscan is the ability to do 3D-processing, if a Z-stack was acquired. For example, use Fast Airyscan, and 2x averaging, to take a Z-stack through the region of interest (ROI). Then, use the "3D" Airyscan processing mode on the whole stack, and the increase of resolution in each individual Z-plane will benefit from information the software gathers from the planes above and below it.