Posted by
James Pawley on
May 14, 2018; 12:03am
URL: http://confocal-microscopy-list.275.s1.nabble.com/confocal-detectors-and-deconvolution-tp7588223p7588253.html
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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
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On May 11, 18, at 10:16 AM, MODEL, MICHAEL <
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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 <
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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)
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