Determine the sampling distance for CLSM

Theory
1

Hi all,

I am currently working on measuring the PSF of the confocal microscope, and then I will use that PSF to deconvolve an image. However, I am not certain with the sampling distance I use.

My equipment are listed below:
-0.1 micrometer latex beads(to measure the PSF)
-473nm light source
-100x,0.8NA Air lens
-Piezo stage to scan the specimen
-Single-photon avalanche diode as the detector

I estimate the resolution of my microscope to be 360nm by using 0.61λ/2NA formula, in order to satisfy the Nyquist theorem, I divide 360nm by 3 which equals to 120nm. So I set 120nm to be the sample the PSF of the latex beads, but I am not sure whether 120nm is small enough to sample the PSF of 0.1micrometer latex beads and deconvolve an image. Is there any suggested article or paper discussing about this problem?

Thank you all for reading.

2

Hi,

First, you want to use 0.61λ/NA for lateral resolution. 0.8 is a very low NA for a 100x magnification. The best resolution you can hope to get is ~400nm, so you don’t need to use 100nm beads. 200nm beads should work better as the increased signal you get from the larger beads will help with the lower NA objective.

SVI’s calculator has been mentioned here numerous times - and is a great place to start to figure out initial settings.

1 Like
3

I made a typo in the formula, thank you for the correction. And your advice is very helpful, thank you!