Dear Confocalists
Following the recent discussion about enhanced PMTs, I would like to ask a
couple of questions and share with you our experience. Coincidently, we had
those PMTs installed last week in our 4 year old MRC-600. Before proceding
to the PMT replacement we collected an image with defined settings and after
installation of the new PMTs, the same image was collected. That's when
things started to go wrong. Since the laser was realigned as well as the
mirrors, I was expecting to obtain the same image quality either with lower
gains and/or less laser power as advertised by Bio-Rad. However, we found
that in order to obtain the same signal intensity the gain settings had to
be INCREASED (from 3.0 to 6.5 in PMT1)(same laser intensity and pinhole
aperture). We then measured the photons/pixels value for the different PMTs
using the Bio-Rad protocol to test the Relative Sensitivity Improvement
(RSI) of enhanced PMTs. With this protocol a RSI of 1.06 was obtained for
PMT1 and 1.02 for PMT2. This means of course that the enhanced PMTs showed
no improvement over the old ones. These numbers can suffer minor changes if
the confocal mirrors are not reajusted after replacing the PMTs.  And here's
my first question. If the new and old PMTs are apparently equal (the RSI is
virtually 1 meaning the photons/pixels are the same in new and old tubes)
why the gain settings had to be more than duplicated to obtain the same
signal ? Furthermore, and under exactly the same settings, "accumulate to
peak" with new PMT1 needed 6 frames while old PMT1 needed only 2 frames. Why
this difference ? Finally, if one has a pair of enhanced PMTs with a RSI=2
(or more) can we really expect to obtain a similar signal if the gain
settings are decreased by a similar factor ?

Thanks very much for any answer on this subject. Kind regards

  Rui





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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]
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