what is your experience for deconvolution of ligh-sheet data.
in particular what is the best way to compute/estimate the PSF.
big topic, many opinions, here’s mine:
In my opinion, the “best” way to measure a PSF used for deconvolution is to (carefully) measure it directly with a bead. But, for the reasons explained below, it’s important that you do this using the exact same sheet (NA, wavelength, etc…) that you use to capture your raw data, and that the sheet be in the detection plane similarly between the PSF and the raw data.
For your other questions, the important thing to remember (and the “model” for it all) is that the effective “system PSF” in a light sheet (indeed, pretty much any microscope) is the product of the excitation PSF (in this case the sheet), and the detection PSF (your “widefield” PSF that you would see with epi illumination).
A good reference for this concept, if you’d like to read more, is Engelbrecht & Stelzer (2006)
Keeping that simple model in mind, the answer to the rest of the questions becomes clearer
For a classic light-sheet with gaussian beam the light-sheet thickness changes and this also changes the PSF?
Yep. The system PSF (the product of the detection and excitation PSFs) will change if you change either the sheet itself or the detection parameters.
does someone of you know why the PSF in a light-sheet looks so different than an EPI? It looks V shaped
That usually means your sheet is a bit out of focus. In that image above, I showed the “widefield PSF” and the light sheet profile both centered in the middle of the image… but that doesn’t happen for free, it requires alignment of the system, and if your light sheet is slightly outside of the detection plane, then the product of your sheet and your detection PSF will be skewed to one side. Here’s some read data I took to demonstrate that concept, shifting the sheet slightly out of focus. (note, the funny additional side-lobes are because this is a lattice not a gaussian sheet)
going back to the question of deconvolution, you can imagine now, that if you collect raw data with your sheet in a different state of “defocus” than the PSF you used for deconvolution, you’ll get artifacts. So, extra care care is required during both acquisition of the PSF and the raw data.