Stupid question about Zeiss Airyscan raw data

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Greetings! An Airyscan user presented me (who have never used it) with processed images showing interesting pattern, but I noticed a bunch of potential sources for artifacts and concerns in the raw Airyscan data. To be able to convince him and myself, I need to confirm the following: if I average the raw stacks acquired on all the Airyscan detector segments, then I’ll obtain a regular confocal stack, right?

Thank you, Zeiss Airyscan experts out there!

-lin

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In short: yes, more or less.

In super-resolution mode, the zoom optics projects an area corresponding to 0.2 Airy untis onto one detector element. The entire detector area corresponds to 1.25 Airy untis, which is a bit larger than the default size of the physical pinhole in regular confocal mode (usually set to 1 Airy unit). The sum of all detector elements gives, thus, an image with a bit less resolution than a regular confocal image.

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Thank you for the answer; that’s exactly what I need to confirm about…

A related question: there’s a second channel “ChA#” in the raw data file that contains just one image stack. What info is in that channel in relation to “ChA”, the Airyscan raw stacks?

Thanks! --lin

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Hello Lin,

for an airy image you have a file 32 stacks. As you correctly saw, the second channel contains also an image. The 1st “plane” is the sum of the 32 airy channels. What is a little bit confusing is that if you sum the channels directly you do not get exactly the same values. The reason is that the sum is done in 16 bit and then converted back to 8-bit.

Overall you can use the 2nd channel to see if your confocal image @ 1.25 Airy-units has a problem or not.

Here the explanation by Zeiss customer service:

Regarding the file format and channels: the first channel are the raw data of the 32 Airyscan detector elements, called phases. The second channel is the sum channel (all 32 8-bit channels summed up in 16-bit or floating bit space, then divided by 4 for useful 8-bit display). Only the first phase has this information. The other 31 phases are empty and only present because of the data format convention (since 32 phases are needed for the first channel).

I hope this helps.

Antonio

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