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Laser power stability -Does it exist in a confocal microscope?

Almost all lasers used in a confocal microscope usually are rated to
have less than 1% power fluctuations when measured at the laser head.
However in the confocal microscope, one can easily obtain over 10-20%
power fluctuations  if it is measured on a stage after the objective or
in the PMTs.
How can 1% stability change into 20% instability. What is causing this
problem?

Recently our system that normally had cyclic fluctuations  between 5 and
25% (since it was delivered new)  developed fluctuations in the order of
100-400% peak to peak variations.   It was impossible to set a top and
bottom threshold  as the power varied greatly in a periodic cyclic
fashion while taking a stack of images .

Solution--After many months of trial and error testing  The AOTF was
replaced. . after the AOTF was PROPERLY  alignment the power
fluctuations were reduced  to less than  5% .   Proper alignment of the
AOTF was the key to reducing the power fluctuations.Replacement without
proper alignment was insufficient to correct the laser fluctuations.

Although improper heat dissipation can greatly effect the laser power
stability, this new data from our system strongly suggests that if a
AOTF is miscalibrated the system  can yield power fluctuations  that
will greatly effect intensity measurements.All confocal systems that use
a  ATOF to separate laser beams may be effected by this AOTF issue. . I
am glad this has been finally solved on our equipment.

The data described above has been accepted  for publication and will be
published in Cytometry this summer with the following title: "Quality
Assessment of Confocal Microscopy Slide Based Systems: Instability" .
PDFs of this paper will be available after publication.

The data will also be discussed later this month in a 2 hour tutorial at
the International Society of Analytical Cytometry (ISAC)  May 20-24
Quebec  City  Canada.
Please contact me if you need additional details on this observation or
the tutorial.
AOTFs are great for confocal microscopy operations but it appears that
they may introduce serious power fluctuations in some systems. You must
check your system to detect 5-20% power variations. However 100%-400%
can be detected by observation of screen intensity.

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 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]