Well, I’m not clear why you are using confocal at all for this application. If Z resolution doesn’t matter surely you will be better in wide field? And I really can’t see how deconvolution will help. Pace various vendors, if you cannot sample at the resolution limit, I don’t think it will give you any more than conventional noise-reduction and sharpening filters.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jan Trnka
Sent: Thursday, 12 August 2010 10:49 PM
To: [hidden email]
Subject: Re: Optical slice thickness and number for PSF and deconvolution
OK, I understand that it's best to sample at Nyquist. However, for my experimental samples (looking at mitochondria in live cells over several hours) this would cause a lot of photodamage and bleaching. Since I don't really care about z-resolution (at this point I basically want a 2D picture of a whole cell with all its mitochondria) I decided to go for a wide pinhole, which allows me to get the picture I want in one go. Maybe this isn't the best way of doing it (any suggestions welcome) but my impression is that such a picture (or a z-stack of thick slices) could be deconvolved to get sharper pictures. Does this make sense? If so, how do I get a PSF for such deconvolution?
On 12 Aug, 2010, at 2:12 PM, Vincent wrote:
*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
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Vincent Schoonderwoert, PhD
Scientific Volume Imaging bv
Hilversum, The Netherlands
[hidden email]
[hidden email]
Tel: + 31 35 646 8216
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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.
Department of Biochemistry
Department of Biochemistry
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