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The mechanical slits used to select spectral regions for detection can
and do fail or change response on a periodic basis.  They are
electromechanical, and with heavy use they wear out and need to be
replaced and properly recalibrated.  It is not always obvious that there
are problems when imaging conventional biological specimens, the biggest
clue is an increase in background signal due to reflected light.  It is
wise to habitually check to make sure they are functioning properly, one
bad slider motor can mess up all the detectors, because they are
interdependant when bandwidth selections are made.
-Karl

Robert Zucker 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 observed 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]
>
>

--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu