Reply from Bio-Rad re: Enhanced Photomultipliers.

Rui Malho asks the straight question `Can we expect a (Bio-Rad) "enhanced PMT"
to have a sensitivity increased by a factor of 2 (or more) at similar voltage
inputs when compared to a "standard PMT".

The answer is yes.

The Bio-Rad enhancer is a prism assembly mounted on the front window of the PMT.
In our MRC-1024 brochure there is a diagram of the enhancer. It works in the
following way.

All photons in the beam are deflected into the photocathode of the detector at a
critically chosen angle. Multiple internal reflections inside the front window
result in multiple interaction with the photocathode (via an evanescent wave)
and a consequent  increase in Quantum Efficiency by a factor of up to two in the
detection range 400nm to 700nm. More photo-electrons per photon leads directly
to the highest quality images. Note that the enhancer only works if the incident
light is nearly parallel, as is the case in the Bio-Rad confocal design.

How do we know that there is an enhancement?  Because we measure the performance
of each individual PMT during assembly both before and after the enhancer is
attached.

The important thing to understand is that the enhancement is in the quantum
efficiency i.e. sensitivity of the PMT. This is what matters. The sensitivity is
almost independent of gain (i.e. tube voltage). This is what you would expect
since the efficiency with which light is converted into electrons is a property
of the photocathode and enhancer (if fitted) and not the voltage applied to the
tube. Because the image gets brighter when you increase the gain does NOT mean
that you are detecting more photons. If you need to convince yourself of this,
image a featureless sample such as fluorescent plastic and measure the signal
and standard deviation of a selected area at different PMT voltages. The ratio
will not change much with voltage.  ( Square the ratio and you have worked out
the absolute number of photons detected, thanks to Poisson statistics.)

One last point.  2 photomultipliers of identical quantum efficiency may require
different operating voltages to produce an image of the same brightness. This is
because the gain of the 2 tubes is different.  However, for the same microscope
settings (laser intensity, confocal aperture etc.) the signal/noise in the image
will be the same with both PMT's and will be broadly independent of the actual
voltage applied.

 I hope that this helps to clarify the situation.


Dr. Andrew Dixon
Scientific Director
Bio-Rad Microscopy Division





______________________________ Reply Separator _________________________________
Subject: Enhanced PMTs (still)
Author:  Confocal Microscopy List <[log in to unmask]> at
Internet
Date:    13/02/97 18:22


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]
______________________________________________________________________