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Deborah,

PMTs when probably adjusted should manage at least several orders of
magnitude of linear response as long as you are not butting up
against PMT's saturation limit, which is a very bad thing if you are.
In general, one would have be very careless to let that happen.

However, linearity problems occur frequently because of other
factors, especially power saturation of the fluorophore. For example,
a 488 nm line even at 0.1 mW power using a 1.4 NA lens on an FITC
labeled sample will show non-linear effects because of the finite
lifetime of the fluorophore. Even at 0.1 mW, some photons delivered
to the sample will not be proportionately absorbed because a
significant fraction of the fluorophores are in the excited state and
can't absorb more light until they emit a photon or decay back to the
ground state by some other path (e.g., intersystem crossing, etc).

If you think about PMTs in general, and consider that most
spectrophotometers and fluorometers use PMTs are linear by 3.5 log
units or better that is equivalent to about 12 bit linearity (1-4096
compared to the 1 to 256 that you were concerned with).

If you need more info, email me direct. Regards,

Mario


>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>When the gain and offset of the detectors are correctly set and no
>averaging is used, is the 0 to 256 dynamic range linear on a confocal
>image?
>
>Many thanks!
>Deborah Wessels, Ph.D.
>W.M. Keck Dynamic Image Analysis Facility
>Room 14 BBE
>Department of Biological Sciences
>University of Iowa
>Iowa City, IA 52245
>
>phone: (319) 335-2883
>FAX: (319) 335-2772


--
________________________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies LLC
President and CTO
ph (510) 528-2400

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