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 in "LSM" Airyscan (as opposed to "Fast" Airyscan):
- 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)
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The system requires calibration
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- 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 ("FAST" button in the left-most panel of the software, next to "LSM") 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
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