Averaging PSF of beads for input to DeconLab2 and FlowDec

Hi all,

I’m relatively new to image analysis. I think I understand the basic of deconvolution, but I’m stuck on how to best put it to practice. I think this should be a simple problem to solve, but the keywords that I’m searching must be incorrect.

Goal: Command line deconvolution of large microscopy volumes using DecovolutionLab2 or FlowDec.

Problem #1: Both programs require a PSF in tiff format. I’m practicing with these software using a generated PSF from PSFgenerator. However, in real life I understand that I will need to generate a PSF specific to my microscope. I plan to do so using this as a guide. To summarize; take a z-stack of many beads sampled identically to my image, extract the PSF of each subresolution bead using MetroloJ, average the FWHM of zy and z.

This is where I am stuck. How do I then convert the average FWHM into a tif file?

Problem #2: FlowDec can also use a dynamic PSF using the below as an input.
{"na": 0.75, "wavelength": 0.425, "size_z": 32, "size_x": 64, "size_y": 64}
I don’t understand how this provides any PSF information for deconvolution. It sounds like a dynamic PSF would be best for a real life data, but I’m not sure what this info in json format means. Is there anything I can read to better understand this approach?

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Hi Andrew

It’s been a couple of months but if you are still working on this problem here is a thread that may be of interest.

Particularly towards the end of the thread we discuss how to extract the PSF and some pitfalls that a user encountered. The most up to date PSF extraction script is here