*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
***********************************************************
Vincent Schoonderwoert, PhD
Scientific Volume Imaging bv
Hilversum, The Netherlands
[hidden email]
[hidden email]
Tel: + 31 35 646 8216
***********************************************************
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
Tel.:
+420 26710 2410