How actually the pixels are created by PMT detectors in confocal microscopes?
I understand till signal amplification by dynodes inside PMT and voltage generation at Anode, but how this voltage is transformed into Pixels is not clear to me.
How Integration and Signaling Analog to Digital Conversion works?
Hi jangra
Confocal have a scanning system (either scanners or spinning disk…), so at each moment intensity on your PMT is related to a X Y position. If you scan at 200µm/s and integrate signal every 10ms then you will have a pixel size of 2µm.
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PMTs detect light, and the signal is amplified and converted to voltage. This voltage is then passed through an Analog-to-Digital Converter (ADC), which turns it into digital values representing pixel intensity. Integration time determines how long the signal is averaged for each pixel, affecting its size and clarity.
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So the integration time is what software shows as Pixel dwell time, right!
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This is a very interesting question, Im also interested in it. It seems that a lot of microscope or detector manurfacturers do more that just simple integration. This becomes even more of a mystery when using newer detectors like the SPAD, GaAsP and hybrid detectors.
This you can notice for instance if you just change the pixel dwell time on your microscope, in most systems this does not increase your signal, so what is happening there, its not just simple integration.
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Well for confocal there can be multiple scanning mode, notably one where you really spend time on each ‘pixel’ position and one when you do a very fast scanning, and spend more time at the border of the field of view (scanners have to turn around), so that you do not get the same exact dwell times on each pixels.
Even with numerical integrations, more dwell time should mean more photon,within a linear process. It is strange that you do not get more signal, but check qunatitatively, at some point with the eye you will not see a difference in signal because noise will be negligible anyway.
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