Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/confocal-detectors-and-deconvolution-tp7588223p7588254.html
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Hi Mike,
Collect two 'as clean as possible' images, deconvolve and compare those.
I have occasionally acquired on our HyDs:
line accumulation: 16
frame accumulation: 16
(both max'd out ... I would like Leica to enable the drop down lists to
go to 100, or even 256, instead of max 16 ... I manage a core that
charges by the hour, so more accum --> more revenue <--- better data).
Pinhole 0.5 Airy Unit (the graphs I have in the SP8 room -- from some
Zeiss pdf -- show some more improvement in XY if go to 0.28 airy unit,
for sparse features this would be ~0.3^2 = 0.09 vs ~0.5^2 = 0.25,
photons getting through the pupil than 1 A.U.
63x/1.4NA objective, 40 nm XY pixel size, 120 nm Z. Modest laser powers
(yes, some photobleaching, usually). I acquire in 16-bit mode even if
doing only a few line accumulation and frame accumulation = 1.
'Standard' fluorophores (i.e. Alexa Fluor 488) and mounting media
(mostly Prolong Gold with the 1.4NA lens). Yes, would be better with
better fluorophore (i.e. Janelia Fluors, maybe BD Brilliants, Thermo's
SuperBrights, more) and optimal R.I. matching per specimen and objective
lens.
* Then try out a couple of deconvolution settings wrt SVI's options in
HyVolution2.
On GaAsP PMT, optimized gain (and offset), optimized choice(s) of
summing and/or averaging (i.e. 16-bit mode).
George
p.s. Allison - great post about us wanting quantitative devices and the
companies should stop pushing 'a pretty picture'. Memo to confocal
companies: if we just wanted pretty pictures, we would be running cRAYon
Distribution cores.
On 5/11/2018 11:16 AM, MODEL, MICHAEL wrote:
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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?
>
> -----Original Message-----
> From: Confocal Microscopy List <
[hidden email]> On Behalf Of Steffen Dietzel
> Sent: Friday, May 11, 2018 10:49 AM
> To:
[hidden email]
> 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
>
> --
> ------------------------------------------------------------
> 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
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>
http://www.bioimaging.bmc.med.uni-muenchen.de--
George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamarahttps://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650http://confocal.jhu.eduJuly 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstractsupporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures