Re: Optical slice thickness and number for PSF and deconvolution

Posted by Vincent Schoonderwoert on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Optical-slice-thickness-and-number-for-PSF-and-deconvolution-tp5415740p5415867.html

*commercial interest*

Dear Jan,

The amount of the signal in images is mostly judged just after image acquisition. Based on this it is often decided to use a wider pinhole.
As you probably know, when deconvolution is properly performed you will gain not only an increase in resolution but also in signal. Therefore, we advise to close the pinhole and use deconvolution for increasing the signal (to noise) before determining the quality of the image.

As with imaging the object of interest it is important to follow the Nyquist criteria for imaging the bead images.
We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to determine these rates. You can also create a picture here of your theoretical PSF to get an idea of its dimensions.

In general it is best to really match the Nyquist criterion in xyz. Else you can go for 2x more. This however may introduce other problems like e.g., bleaching. If the bead images are differently sampled it requires interpolation for matching that, making the process of deconvolution more computationally demanding. Thus Nyquist is okay. Another important thing to keep in mind is that you need to image enough planes to cover your PSF.

I hope this answers your questions.
Best regards,
Vincent


Jan Trnka wrote:

Dear list,

this is probably a trivial question but so far I haven't found a good answer. When taking 3D images of subresolution beads in a confocal microscope (for PSF construction) does the number and thickness of slices in the z-stack need to be exactly the same as that of a sample to be deconvolved? I understand the x-y dimensions need to be the same but how does it work for z? Would a higher number of thinner slices (finer z resolution) of the bead improve the construction of the PSF? My actual samples are imaged with a rather wide pinhole setting to limit the exposure of the sample (live cells) and thus provide quite thick optical sections.

Thanks,

Jan

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

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