Question about three parameters to estimate PSF with 'Diffraction PSF 3D' plugin

psf
deconvolution
plugin
Tags: #<Tag:0x00007fb882ceb610> #<Tag:0x00007fb882ceb2f0> #<Tag:0x00007fb882ceb0c0>

#1

Hello everyone,

This post is to see if someone could help me to clarify three input parameters of the Diffraction PSF 3D Plugin (longitudinal spherical aberration, image pixel spacing and slice spacing (z)). Below are some details:

I am trying to use the Diffraction PSF 3D and create an estimated PSF for the Images I am working with, which are 3D images obtained with a Confocal Laser Scanning Microscope.

On the ‘Specify PSF’ window, there are some parameters I need to set. I tried to read through some documentation (https://imagej.net/Deconvolution http://www.optinav.info/Diffraction-PSF-3D.htm) but I am not sure of what to input in three parameters:

  • Longitudinal spherical aberration at max. aperture, same units
  • Image pixel spacing, same units (ccd cell spacing / magification) (I used 20 initially, 20x lens though I am not sure of it)
  • slice spacing (z), same units ( Also used 20 initially)

The screenshot of the “Specify PSF window” has the parameters of the lenses and medium (NA 0.75, IR 1.000, Magnification 20x) and excitation wavelength (the sample used eGFP with lambda 488 nm)

I understand that the longitudinal spherical aberration is the type of aberration that affects the light distribution in the axial direction. Unfortunately, I am not sure what is the proper way to calculate it or if I have the information that I need to calculate it.

image

I really appreciate if someone who knows more about deconvolution could help me on this. In case additional information is needed I will provide what I can as fast as I can.

Thanks!!
Cheers,


#2

Hi @leandroscholz

I don’t know this plugin in detail. I am not sure how Longitudinal Spherical Aberration is computed.
I might be able to help you with a couple of things… (I am a programmer/signal processing guy, not a microscopist, so a good microscopist may be able to give you better advice on filling out the scope parameters)

  1. Image Pixel Spacing, is the image pixel size (usually between 100 and a few hundred nanometers)
  2. Slice spacing is the distance between z-planes, ussually a few hundred nanometers, most often it is ~2-3 times bigger than the pixel spacing. That’s because lateral resolution is better than axial, and the slice spacing is chosen based on resolution.
  3. The above two parameters might be in the meta data of your image
  4. Does this plugin have a “confocal” mode?? I don’t see one. If it is a widefield PSF you will have to square the PSF to use it with a confocal image.
  5. You also might want to look at the Big Lab PSF plugin. It has a Gibson Lanni model, and the parameters that determine the SA in the Gibson Lanni model are more intuitive (sample refractive index, and distance from coverslip). I don’t think PSF Generator has a confocal modality, so again you’d have to square it, to use with a confocal image.

#3

@bnortham Thanks for the reply! So, in the end, some of the terms (1 and 2) from your answers were much simpler than I thought. They just used names I didn’t know were exatcly synonyms.

I don’t think so.

This plugin option seems to be more complete. I will look at it.
What I will probably do is obtain a PSF and then use the iterative deconvolve 3D plugin.

I have an additional question: When you say square it, what operation do you mean? (Sorry if the question is too simple).

Thanks again!


#4

Multiply the PSF by itself see this thread Richardson Lucy TV deconvolution

Also you may want to try a measured PSF using beads. Deconvolution from true psf with beads

There is also some deconvolution utilties in image-scripting, they appear under Templates->Deconvolution on the script editor. I added a script to extract the measured PSF from beads but I’m not sure if that is being distributed yet (@ctrueden when was the last time imagej-scripting was released?).

I also recommend DeconvolutionLab2. Which I haven’t actually used myself, but I used Deoncolution Lab 1, and it was good, so the sequel must be better right??


#5

Check out this page: http://status.scijava.org/

It shows the release status of all components. The imagej-scripting component was last released 2017-05-24@16:09:49, and last updated 2017-10-05@23:29:16, and its current status is “Cut” which means a new release needs to be created to bring things back up to date with the current master branch.

Actually, due to the recent Travis updates, nearly all components now need a new release to make that page happy.

I am cutting release 0.7.0 of imagej-scripting now. Stay tuned! Release 0.7.0 of imagej-scripting now exists! @bnorthan Henceforth, it is now possible for you to cut your own releases of this (or any) component by using the release-version.sh script as described on the wiki.

Note that the status.scijava.org page does not update automatically after every release, but only nightly. And actually at the moment, not even nightly—I still need to hook it up to a Travis nightly cron job.