Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185p5350837.html
Hi Sandrine,
Detector gain (PMT voltage) - ideally would like to be a modest
value, such as 600 or 700.
Amplifier offset - adjust so that no laser light results in all
values being above zero. Note that if you change scan speed and/or
detector gain, the offset value(s) may change. When using 12-bit data
mode, I typically aim for an offset of ~100, but am happy with
anything less than 200 (at moderate gain).
Amplifier gain - you are correct. Do not change from 1.0. It is just
does post acquisition arithmetic.
Other items:
You did not mention what fluorophore(s) or fluorescent protein you
are using. Or whether you are using the best filter or other
wavelength setting(s) for these. Depending on the cell's
autofluorescence, this may need optimization.
You did not mention laser power:
* Make sure the laser output is stable! Both within a frame and over
experiment time. A starting point is to turn on the transmitted light
detector (which as a bonus gets you the cell morphology) and see that
you are getting stable intensity. I recommend no polarizing elements
in the light path. If you keep your confocal system off at night, may
take many hours for the laser output to stabilize (I have never been
sure whether it is the lasers or the AOTF intensity controller as
being the guilty party or parties).
* If you are using an argon visible laser on an LSM510, run the laser
at 6.1 Amps and leave the laser power dialog open (it might fluctuate
on its own between 5.9 and 6.3 Amps - that is ok). On our system I've
seen the power change to crazy values, like 7.9 Amps, apparently on
its own (also on hitting re-use, even though I know the saved setting
was not that value). On the LSM710 Argon laser's remote, turn the
knob so it is in the middle of the green light range.
* Laser power to the specimen ... lower is better! For example,
cardiomyocytes die a spectacular death after a few seconds of 488 nm
excitation when loaded with Fluo-4 and imaged at high power.
If you are using an -AM conjugate, more is not better. Besides the
possibility of turning your Calcium ion reporter into a chelator, the
-AM leaving group is formaldehyde. Could be worse: GFP maturation
produces one molecule of H2O2 (so two for CaMeleon), and red
fluorescent protein maturation may produce two H2O2's.
No pixel in the background at zero ... correct, this is ideal.
However, at high gain (say 900 volts) and an offset such that no
laser light results in average intensity value around 100 (12-bit
mode), when you are imaging with the laser and specimen, if you
acquire enough images, eventually statistics dictate that some
outlier will reach zero. So what! When you use the "no light" image
(or an average of a whole bunch of them) in background subtraction,
lots of values outside the cells will hit zero.
A more important item is getting the detector gain and laser power
optimized so you can use most of the dynamic range of the confocal to
get the data you need. This requires guesswork or using the first
specimen to get maximum intensity (typically, saturated Calcium ion
with ionophore).
Best wishes,
George
At 09:05 AM 7/28/2010, you wrote:
>Hello.
>I had this discussion with several colleagues (biologist like me),
>and did some
>research on my own, but I figurate the best would be to submit the question
>to this list. Here's the point: we are doing quantitative measurement with
>confocal microscopy (calcium measurement) using a Zeiss LSM exciter. There
>are 3 parameters of the PMT that can be configured: detector gain, amplifier
>offset, amplifier gain. The only parameter I adjust to improve the
>signal is the
>detector gain. I keep the amplifier gain at 1 as I read in several
>papers that
>increasing it will not improve the signal over noise ratio (they
>also say that it
>is bad for several reasons that I can not summarize here). Is that correct? I
>put the offset usually at zero. I saw that a change in offset can affect the
>calcium signal. In any case, I keep the same offset in a series of
>records that
>I whish to compare. Any comment on this? My guess is that the same should
>be done with the amplifier gain.
>Ah, a last point: the offset should be tuned so no pixel in the
>background has
>the value of zero right?
>Does anybody have any clarification for these questions? Thanks for your help.
>
>Regards
>Sandrine
George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+
spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara