[this is a copy of a post originally posted in image.sc]
Hello,
I am a PhD student, and recently begun to use the confocal microscope (LSM710) with Airyscan, and I have some doubts/curiosities—hopefully, you can help me out:
- The first question is related to the detector: I notice in Zen that the files after the acquisition have below the z-position a slider for “phases” and there are 32-phases; what are they, what they represent from the actual image? I imagine that they would have data collected from the sample (therefore they represent the 32 honeycomb detectors), but if that is the case why can it be displayed (the image displayed is black)? This is intriguing to me because only when I have phase-1 it’s when I can see an image of my sample.
- Second and third questions are related to each other: what is the correct way to look at airyscan data to then analyze: acquired the raw airyscan super-resolution data and go with it (analyze in ImageJ or CellProfiller), or the file needs to always be pre-processed in ZenBlack to be analyzed? Using the latter I noticed that my images are reduced to ~4 mb from 130 mb, I am curious what kind of compression is done here that guarantees no data loss (or if its just the case that data is allocated but nothing is stored in the raw file?)
- a. Also, is there another way to process airyscan data besides Zen black?
- If I acquire images with Airyscan super-resolution and perform the “processing airyscan” can I compared intensities between my samples? When the acquisition settings are the same. Is this fundamentally wrong? Aren’t the detectors measuring photons in a comparable way to a the T-PMT detector?
- What is the guideline when using airy scan super-resolution and the thickness of focus is different between two channels: e.g. green is 0.42 µm and DAPI is 0.38 µm (arbitrary numbers for these example), is it ok to use different thickness or they should be matched?
A bit of context: in my experiment I am trying to count foci of a target protein in the nuclei of cells, using a pipeline in CellProfiller to segment and measure the objects in the images; but, in case that does not work well I was thinking if instead the intensities could be compared. But given the fact that the there’s a lot about the Airyscan detector and data processing that I don’t know, I am not sure if that is right to do.
Lastly, what do people mean when they say that airyscan super-resolution quote: “is not confocal”?
Thank you very much for your time.
cheers,
leo
