Just one comment related to this. When running in light mode, the laser would
adjust current in order to have constant No. of photons output. This means that
a few % variation in current might not be translated into equivalent light
variation.
All the best,
Nuno Moreno, PhD
Head of Equipment Management
Instituto Gulbenkian de Ciência
Fundação Calouste Gulbenkian
Tel . +351 4464538
Fax. +351 4407970
On Jul 29, 2010, at 2:55 AM, George McNamara wrote:
> 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