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
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