Deconvolving Spinning Disk Images

Posted by Dani Bodor on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolving-Spinning-Disk-Images-tp6508446.html

Hi there,

I would like to deconvolve my spinning disk images for quantification purposes. Regarding this, I have a few issues/questions.

1. I have been trying to image fluorescent beads, in order to obtain PSF measurements for my microscope (conditions). However, whenever I do this, I always “see the grid” of the spinning disk unit, meaning that there are multiple points visible at the same time, even when a single bead is imaged. This only becomes obvious after severe rescaling and the other grid dots have a very weak intensity compared to the main dot (at least 2 orders of magnitude). I have tried synchronizing imaging time with spinning disk speed, long/short exposures, high/low laser intensities, with/without EM gain, etc. I always see the same thing.
The questions are: Will this affect the deconvolution efficiency/accuracy? Does anyone have any solution for this problem?



2. I have read conflicting opinions on performing deconvolution using experimental vs. theoretical PSFs vs maximum likelihood estimations (MLE). Some say that when imaging thick samples (in my case ~30um) it is better to do MLEs, because the PSF changes depending on the depth that you image, and measured PSFs are always on the surface of the slide. Others say that measured PSFs will always (usually) be better as they don’t assume anything but actually measure what is going on. Then again, I have been considering doing theoretical PSFs, as I have been having a lot of trouble actually measuring a proper PSF in my microscope (see point 1).
Does anyone have any experience with this or can advise me on these issues. Which programs/algorithms/plugins could I best use for this?