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Our confocal is very stable.
We have a SP1 (one of the first ones), used by all the biology department,
without any "real" control (means no technician or scientist really
responsible for the instrument, used by many different groups).
Our group regularly did spectra with 488nm exication and PMT1 for detection
530-730nm, and also with 2-photon excication (detection 400-700nm). We
often "oversample" spectra, to have better signal and a good spectral
resolution (e.g. 10 nm width with 5 or 7nm steps). But we also use 5 or 7
nm width with same step sizing. Depends on the samples.
So, I cannot answer about the different PMTs, but the many spectra we
recorded over 2 years were all OK. (Although we probably would NOT have
detected a 5nm shift, since we assume this to be in the range of error
(temperature changes, physiological changes - we did many in vivo
measurements of cyanobacterial pigments, pH changes in slightly buffered
material, etc.).)
So, we e.g. have the pigment-fluorescence spectra as references, no
calibration lamp or so. But these spectra are originating from fluorescence
of 2-4 different pigments, and show 2-3 peaks (dependent on the sample)
between 450-680 nm, so well suited to compare. Since the spectra are mixed
ones (and they change in different developmental stages) we also did
spectral unmixing by iterative approaches, using the individual (known)
spectra of the different pigments. The fits worked very well, so the
spectra are OK.
Our SP1 was running perfectly for 5 years "as delivered", without
realignment. Now it only was readjusted after changing the lasers, and
again everything is OK.
We of course work at 23°C all the time. We also only used water immersion
lenses (20x and 63x).
Arthur
At 19:39 16.08.2004, you wrote:
>Confocal Users
>How stable is the alignment in your confocal microscopy system? Does the
>beam wander or are the mechanical components affected by heat?
>
>We Tested our Leica SP1 spectral system with a LightForm lamp as
>described our MM 2004 abstract and in a tutorial review on spectroscopic
>imaging to be published in Cytometry (in press) “ Calibration and
>Validation of Confocal Imaging System.” The test measures spectral
>registration of defined peaks between 400-650 using the LightForm
>Spectral Lamp.
>
>Morning: The system showed that PMT 1and PMT 2 were excellent, with
>sharp narrow wavelength reference spectra peaks. PMT 3 had less
>resolution than the other two for an unknown reason.
>Late Afternoon: Strange image data recorded in PMT 2 below the
>647-excitation line while measuring TOPRO-3 (PMT3) and Alexa 568 (PMT 2)
>-Possible reflections were occurring in the detecting region assigned to
>PMT 2.
>
>We next tested the three PMTs in system with LightForm lamp. It was
>observed that PMT 1 and PMT 3 were identical to the morning values. PMT
>2 shifted 5nm to lower wavelength values and the FWHM of the peaks were
>now very broad. This suggests that resolution was lost using this PMT 2
>(our best PMT in the morning). We put sliders over the laser line and
>found the unusable range below the laser lines to be 8nm (488) 16nm(568)
>and 32nm (647). They supposedly should be 7nm or less.
>
>How is this occurring? Why is the system going out of calibration? Must
>we test at multiple times in the day to insure a calibrated system? How
>would you test for laser or machine drift on your confocal machine? It
>is a shocking observation that other confocal users need to be aware of,
>as the ramifications are very serious for good reproducible data. Let’s
>discuss this confocal instability on this user group.
>
>PS—Same effect observeed about 5 different times on our machine over a
>2-year period and at least twice on another machine located in a
>different geographical area. We do not check for this problem all the
>time. Do You?
>
>Best wishes
>Bob
>
>Robert M. Zucker, PhD
>U.S. Environmental Protection Agency
>Office of Research and Development
>National Health and Environmental Effects Research Laboratory
>Reproductive Toxicology Division, MD 72
>Research Triangle Park, North Carolina, 27711
>Tel: 919-541-1585; fax 919-541-4017
>e-mail: [log in to unmask]
Dr. Arthur Schuessler
Institute of Botany, FB10, TU Darmstadt
Schnittspahnstrasse 10
D-64287 Darmstadt
GERMANY
Fax: ++49 1212 66151 164568
e-mail: [log in to unmask]
http://www.geosiphon.de/
http://amf-phylogeny.com/
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