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.
• 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)

Image Added

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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