Seitenhistorie

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.

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• 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.