Posted by
Zdenek Svindrych-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/confocal-detectors-and-deconvolution-tp7588223p7588257.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call them),
note that their dark noise is some 3 - 4 orders of magnitude higher than
that of regular PMTs! Of course, chilling the detector to -80 degC (which is
common with EMCCDs, for example) would solve this issue, but the cost would
be prohibitive...
For more details on SiPMs vs PMTs see here:
https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MBCheers, zdenek
---------- Původní e-mail ----------
Od: JAMES B PAWLEY <
[hidden email]>
Komu:
[hidden email]
Datum: 14. 5. 2018 0:27:35
Předmět: Re: confocal detectors and deconvolution
"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
Hi all,
I would like to Eecho Michael’s points.
Because hybrid photodetectors have a very high gain (>>1,000x) in their
first stage, they produce very little “excess noise” (also called
Multiplicative Noise). As a result, it is possible to characterize their
output as "25 photons detected” (although it might be safer to think of it
as "25 photons detected this time" or ”25 +/- 5 photons.").
Straight PMTs do not share this feature and because single photoelectrons
produce output pulses that vary significantly in size, even the very best
PMTs produce an uncertainty in the magnitude of the signal presented to the
ADC that is at least 40% larger in relative terms than would be the case in
the absence of this excess noise. On PMTs having electron multipliers
optimized for other reasons (such as making them very small, like those in
the 32-PMT linear arrays), the increase in uncertainty is closer to 100% (i.
e., The signal has the same uncertainty that it would have if 4 times fewer
photons were counted perfectly.)
Either type of PMT can have a GaAsP photocathode but it will need to be
cooled.
Although single APDs may have a high photon detection efficiency (PDE, a
spec that is like QE but which includes the signal lost by photoelectrons
that do not avalanche at all) they have such massive excess noise that it is
essential to use them with pulse-counting circuits and these circuits are
just too slow for use in beam-scanning light microscopy.
The solution is the multi-pixel photon counter (MPPC, a development from the
SPAD (single photon avalanche device),
https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdfhttps://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_ultimate_photosensor/index.html ).
The surface of an MPPC is covered with an array of 400 to 20,000 APDs, each
connected to the high-voltage rail through its own damping resister. The
resistor causes the voltage across the APD to drop as the avalanche
proceeds. This quenches the discharge and produces single-photon pulses of
very uniform size. As all the APDs are electrically in parallel, these
single-photon current pulses simply add up producing an output current
signal almost devoid of excess noise. What could be better? And in addition
they are significantly less sensitive than hybrid PMTs to overheating damage
if accidentally exposed to a bright light.
There are of course limitations: 1) A significant fraction (20-40%) of the
MPPC's surface is taken up with the resistors and the wiring to provide each
APD with + and - voltages. Photons absorbed or reflected in these areas are
lost. 2) The system is only free of pulse-pileup losses to the extent that
no APD absorbs more than one photon within its RC relaxation time (set by
the R of the resistor, and the capacitance (C) of the sensitive area of the
APD. Larger individual APDs “waste” proportionally fewer photons hitting the
resistor and wiring, (increasing their effective QE) but this increases
their capacitance (making them more susceptible to pulse-pileup).
All will be well as long as the number of photons absorbed in the active
areas during time period RC is small (5%?) compared to the number of APDs in
the array THAT ARE ILLUMINATED BY THE BEAM.
The caps above are to remind everyone that, to work properly, the size of
the ray bundle striking the MPPC must be matched to the size of the APD
array (1.3 to 6 mm square). This can be a problem if we imagine the ray
bundle being limited by a confocal aperture that can be varied in size over
a substantial range. (Do we need a zoom lens to make all possible signal ray
-bundles match the size of the MPPC array?)
Apart from this, I would like to second the comment that deep imaging is
usually limited by spherical aberration and systems that can correct for
this without “bumping the specimen while you try to adjust the collar” are
to be preferred.
I would also like to reaffirm that, assuming the pixel size meets Nyquist,
you should ONLY evaluate results after deconvolving the data with an
appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist-
sampled image will be at least 4 (more likely 5) pixels wide, all the noise
terms affect single pixel values. i.e., they have frequency components at
least 4x higher than that which can represent any real structure. In scanned
fluorescent imaging, one deconvolves more to reduce noise than to increase “
spatial resolution” (although you can also increase resolution as long as
you have massive amounts of signal.)
Cheers,
Jim Pawley
****************************************
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
Canada, V0N3A0,
Phone 604-885-0840, email <
[hidden email]<mailto:
[hidden email]>>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
On May 11, 18, at 10:16 AM, MODEL, MICHAEL <
[hidden email]<mailto:mmodel@
KENT.EDU>> wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
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
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
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
http://www.bioimaging.bmc.med.uni-muenchen.de"