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We have potential users who want to quantify some kind of small aggregates in the brain. I am afraid that deconvolution can make noise look like such aggregates. Perhaps collecting a noisy image twice and comparing two deconvolved images might help, but that seems too much work. Am I wrong?
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From: Confocal Microscopy List <
[hidden email]> On Behalf Of Steffen Dietzel
Sent: Friday, May 11, 2018 10:49 AM
To:
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Subject: Re: confocal detectors and deconvolution
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Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
> there is no big difference between HyDs and GaAsP detectors.
I disagree on this. In my view, the major difference is that the HyD always operates in photon counting mode whether, as far as I know, the PMTs (with or without GaAsP) create an electron cloud of which the size is determined by the number of photoelectrons AND statistics, and the cloud size is then digitized. So the output created by one photon may vary substantially depending on the number of electrons created on the first dynodes (which in turn is a statistical process). My information may be outdated and newer PMTs might have extra tricks, if so please correct me.
Another difference is apparently the size of the photcathode. If memory serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs) creates more dark noise. I like our HyDs a lot, I appreciate having a gray value of "21 photons" instead of some random number. But having said this, at the end of the day what counts is the sensitivity of the whole system, and not of the detector alone. So to do this right there is no substitute for testing your own samples on different machines with your applications in mind.
As for deconvolution, yes, it can create artefacts. But so does confocal microscopy (a point becomes an Airy pattern, not a point). And if you do it right the deconvolved image will be closer to the truth than the original image. Should you have the third edition of the handbook around, have a look at the preface, last paragraph.
Steffen
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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging
Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany
http://www.bioimaging.bmc.med.uni-muenchen.de