Well, first of all I would like to thank all who already contributed to this
topic.
It's been quite helpfull. Now for some more comments:

Carlos Rubbi wrote:
>However, in the Zeiss LSM 410, PMT voltage and amplifier gain can be
>controlled separately (both from software). With a constant PMT voltage,
>(and a properly adjusted dark current, called "brightness") image
>intensity varies linearly with the gain. By collecting images at different
>amplifier gains, one can thus virtually expand the 0-255 integer range
>just by correcting for the change in gain.

If this is so, it is an important difference between the Bio-Rad
and the Zeiss (although I had the idea that practically speaking, the gain
controls the brightness of the image). In the Bio-Rad, the gain controls
the voltage applied to the PMTs. And as far as I understood from
 this discussion different PMTs can reach maximum sensitivity at different
input voltage (gain). In this case, what I expect from an "enhanced PMT" is
one that reaches the highest sensitivity at a lower voltage input. Otherwise
I have to increase the gain and, as we all know, above a certain gain the
S/N ratio starts to decrease.

Jim Pawley wrote:
>I can never understnad why, when anyone wants to measure the detector
>system in a confocal microscope they always start by looking at some sort
>of biological specimen.  Although they may be pretty and even interesting,
>such specimens are far from ideal as sources of known brightness.

Well, I don't know if anyone tried to measure the detector sensitivity with a
biological specimen. I certainly didn't. The point it was raised here is that
ultimately, I have to work with live biological specimens and it's the
equipment which has to adapt to the cells and not the opposite. Otherwise,
it's just a useless and expensive piece of machinery. But indeed we measured
(with several non-biological samples) the histogram standard deviation, the
histogram average, the photons/pixels and the RSI. All by the (hand)book.
That was helpfull to estimate if a PMT was more sensitive than other but
does not help much when it comes to the biological work I have to do.
In page 336 of the handbook, Terasaki and Dailey say "We typically started
with a gain setting of 800 to balance image brightness versus noise but we often
decided to use higher gain settings. The lower gain setting produces a less
noisy image but it requires higher light intensity to produce an image of
equivalent brightness." And this precisely the problem I face in everyday's
work: high gain settings and low light intensity. Now, if one can't use a
higher light intensity (because the cells die) then, one has to rely on the
voltage
applied to the PMT. And if an "enhanced PMT" needs a gain setting of 650
to produce the same brightness that an old PMT could reach at 300 (in a scale
from 0 to 1000) then I'm afraid they're quite useless. So probably I should
rephrase my initial question to the following : can we expect an "enhanced PMT"
to have a sensitivity increased by a factor of 2 (or more) at similar
voltage inputs
when compared to a "standard PMT" ?

Cheers

 Rui







_______________________________________________________________________
Rui M. Malho'
Dept. Biologia Vegetal, Fac. Ciencias Lisboa
Ed. C2, Campo Grande, P-1700 LISBOA, PORTUGAL
t. + 351.1.7500069   fax + 351.1.7500048
e.mail  [log in to unmask]
______________________________________________________________________