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Hi Bob,
I am just leaving for hollidays ... so will not get any answers.
I can only say that we had no obvious problems, but as I wrote, we always
used the same PMT. Slight shifts (5 nm range) we would not have detected,
with our biological samples. We often used settings suitable for in vivo
measurements over longer times, so we had to adjust to low laser power, and
often used 10 nm width, with 5 nm steps, as I wrote. We measured the power
at object level with a photodiode, but I don't have the value handy, now.
We also see laser power variation, just by recording changing fluorescence
intensity of a sample over time. In so far, I agree that this should be
checked, but since we anyway cannot make it more stable ...
I wonder why no real approach is used by Leica (and others?) to regulate
and stabilize the laser power. Should be easy by using the AOTFs? At least
the changes we saw were not too fast not to be corrected, I think.
But as I said - our SP1 has no service contract and works quite well !!
And: we didn't make pretty pictures in the noted project, we measured many,
many spectra, mainly in vivo ...
I also know the Olympus/Zeiss/Leica comparison (I mean that one with the
Leica before and after visit of technician, I have a pdf-file). I really
wondered (wasn't it a SP2 ? don't remember) how the shown extremely (I
would say) bad spectra (before re-adjustment by Leica stuff) were recorded
- ours look like those after the tech-visit since long. I think there was
something VERY seriosly wrong with your machine? Were the spectra "before
re-adjustment" just caused by a completely missaligned system?
We unfortunately do not have the money to buy a Lightform, although this
for sure makes sense. But I also never felt a realy need for it, yet.
Arthur
At 22:34 17.08.2004, you wrote:
>HI Arthur
>You must have a fantastic machine for it to work 5 years without
>problems. What is your secret??
>
>How are you measuring stability of your machine? Is it subjective or
>have you applied a specific test procedure to insure that it is stable.
>Most biological samples have too broad of a spectra to be accurate. We
>have had laser power drifts in excess of 25% and spectral shifts of in
>the range of 5-20nm. This can only be detected by measuring the system
>with a specific test procedure. If you do not QA the machine then your
>endpoint of a pretty picture becomes the gold standard. When excessive
>noise comes into the system, it can be observed as bad images. Most
>problems can be masked with extra averaging to remove the noise. If you
>set large detection regions I do not think you will observe spectral
>instability properly.
>
>We have developed a procedure using a Lightform lamp in which fixed
>wavelengths of light are measured across the spectra. These peaks should
>come into the same area on ALL spectral imagine machines including
>confocal microscopes of Leica, Zeiss and Olympus. If you do not test
>for spectral accuracy there is no assurance that the machine is
>measuring spectra correctly. Do you have a test ? It also appears that
>other machine problems can be detected when we observe bad spectra from
>the lamp that may indicate problems with the machine alignment . all
>scientists using spectral imaging systems should consider this Lightform
>test system.
>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]
>
>
>
>|---------+-------------------------------->
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> | Subject: Re: Confocal Instability-shocking
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>
>
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>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) â Calibration and
> >V>Validation of Confocal Imaging System.â The test meaasures 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.
>Letâs
> >discuss this confonfocal instability on this user group.
> >
> >PSSame effecct 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/
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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