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.

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,