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fish-quant / big-fish-examples

Notebooks to illustrate how BigFISH is working.
BSD 3-Clause "New" or "Revised" License
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How to calculate PSF parameters? #7

Closed dakota-hawkins closed 2 years ago

dakota-hawkins commented 2 years ago

Hi there!

Thanks for the great package -- I've been playing around with it for some new smFISH data, and it's been really great and intuitive so far. One question I had, is if you could provide any information on the best way to calculate psf_z and psf_yx from microscope metadata files?

While I've done some experience in image analysis, point spread functions are new to me, so any guidance would be greatly appreciated!

Thanks,

Dakota

muellerflorian commented 2 years ago

Hi Dakota,

this corresponds to the size of the point-spread-function (PSF). Essentially, the size of an object smaller than the resolution limit of the microscope. we use this to set a few parameters, e.g. the size of the filters, in the background.

one fast way to get it (roughly), is to open one of your FISH images and you simply visually check how "wide" your spots are in xy and z, the sigma is roughly half of their width. on a standard widefield system, this will be around 200nm in xy, and 500nm in z.

hope this helps!

best Florian

dakota-hawkins commented 2 years ago

Thanks so much! Appreciate your insight on this. Very much appreciate the reply + the library itself!

muellerflorian commented 2 years ago

with pleasure!