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Hi Max,
The HyDs are fast photon counting detectors. If you use them in "Photon
Counting" mode, your histogram will be perfectly smooth with no missing
values. But in "Standard" mode the photon counts are multiplied by some
constant (e.g. about 100x at 12-bit, 400Hz, 100% "gain") so they appear
brighter. Then your histogram has large gaps (a hundred of zero values
between two big numbers in your histogram, assuming bin width is 1). You may
also note that the signal saturates (intensity value of 4095 for 12-bit
range) at about 40 photons per pixel (at the above conditions). That's why
the "Photon Counting" images are so dim... The reason behind that high
multiplication factor is to ensure the detectors' behavior is linear (Leica
claims the detectors are linear up to 6*10^7 photons/s and in "Standard"
mode up to 3*10^8 photons/s - there is be some correction, although I haven'
t been able to seen it in the histograms of my images)...
In normal PMTs (in "analog integrating" mode), the sharp peaks corresponding
to 1 photon/pixel, 2 photons/pixel, etc., are smeared by multiplication
noise, amplifier noise and slow amplifier response so you cannot distinguish
them.
I can't see any reason why HyD images should be less suitable for
colocalization analysis.
Best, zdenek
---------- Původní zpráva ----------
Od: Max Garre' <[log in to unmask]>
Komu: [log in to unmask]
Datum: 12. 11. 2015 3:10:29
Předmět: Re: Colocalization and Hybrid Detectors
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Hi Neil,
in my hands and on the SP5 we have in our facility I always had some doubts
using the HyD for colocalization: when I analyze the scatter plot in the
colocalization analisys of the Leica software, the pixels of the HyD channel
do not have an even distribution; they are concentrated only in certain
values of the full bit range. I don't know if this is due to the different
gain of the Hyd (used in standard mode) but I feel this effect could affect
the standard colocalization factors that you calculate (Perasons Overlap, ..
.).
Anyway I never tried a PMT vs HyD direct comparison.
My two cents
Max
On Wed, 11 Nov 2015 01:59:29 +0000, Anthony, Neil <[log in to unmask]> wrote:
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>
>Hi all, I hope the scopes are treating you well.
>
>I wonder if anybody has any thoughts on using Leica HyD detectors for
colocalization. If one was forced to colocalize a HyD channel to a PMT
channel, how would this affect the results when compared to a PMT-PMT coloc?
I'm assuming here that both channels would have the same bit-depth.
>
>Thanks in advance for your time.
>
>Neil
>
>
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