Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I agree with Ian Dobbie on this. Most confocals in
normal operation simply integrate the current passing
through the PMT (for example by charging a capacitor)
so that you end up with a voltage proportional to the
number of ELECTRONS which have passed through the PMT.
This will, one hopes, be linearly porportional to the
number of photons hitting the photocathode, and in most
circumstances it is, of course. So the integration time
and dwell time should be pretty much the same.
Photon counting is different. In this case one sets a
threshhold, and any pulse which exceeds this value will
be counted as one photon, while lower ones will be
ignored. In this case one is operating at the fastest
response time the system will give, and the photon rate
must be low enough so that there will not be significant
'pile-up' - ie 2 photons arriving within the response
time. So it's not useful for bright samples. This
seems to be what Julio was thinking of. If the Zeiss 510
does offer photon counting, you will have to find the response
time from the manuals or the manufacturer. But this is
not the normal confocal detection mode.
Guy
Julio Vazquez wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Greetings!
>
> I have a general question about PMT function on confocal microscopes. On
> our Zeiss LSM 510, as an example, the smallest pixel time seems to be
> 1.6 microsecond per pixel for a 512x512 image, and 0.64 microns at
> 2048x2048 pixel size (i.e. not necessarily proportional)
>
> My understanding is that PMT's have a given integration time, typically
> a fraction of the pixel dwell time, during which current pulses
> (corresponding to photons striking the PMT) are counted. Is this correct?
>
> Secondly, is the photon read (pixel intensity on the image) the average
> of the number of integrations for the duration of the pixel dwell time?
> that is, if the integration time is, for instance, 0.4 microseconds, for
> a pixel time of 1.6 microseconds, do I average the four 0.4-microsecond
> values (as opposed to adding the four values)? (if I increase the pixel
> dwell time, my pixel intensities do not change, but I get some reduction
> of noise)
>
> If that were the case, should I expect the same effect on noise
> reduction if I double the pixel dwell time or if I average two images
> collected at halt the pixel dwell time?
>
> I guess the bottom line is, how do I determine the actual time during
> which I am counting photons on a confocal? Does it just depend on the
> particular type/model of PMT, or is it determined by how the software is
> reading and integrating the data?
>
> Many thanks!
>
> Julio.
>
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA 98109-1024
>
>
> [log in to unmask] <mailto:[log in to unmask]>
> http://www.fhcrc.org/
>
>
>
--
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
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