Our confocal spectral imaging systems was tested with an inexpensive
multi-ion discharge lamp (MIDL) that contains Hg+, Ar+ and inorganic
fluorophores that emits distinct, stable spectral features.We have found
that the MIDL characterization verifies not only the accuracy of the
confocal system but the consistency of the confocal spectral imaging
system. The pattern of the lamp shows positional accuracy of spectral
peaks. The FWHM of the peaks appears to represents not only the the
accuracy of the system but also  can be related to the proper alignment
of the system. We find that this lamp is invaluable for accessing the
performance and reliability of all confocal spectral imaging systems.

Recently we found some interesting data that is in need of an
explanation from the participants of confocal list server group.

DATA:  In our system PMT 1, 2 show superior patterns of the MIDL
spectrum compared to PMT 3 with a pinhole setting equivalent to an airy
disc of 1. (10 x lens).  Increasing the pinhole size usually degrades
the spectral pattern (PMT 1, 2).  However,  we found that by opening up
the pinhole to a setting equivalent to an airy disc of 3 and using PMT 3
actually increased (not decreased) the resolution of the MIDL spectral
pattern. The FWHM of the MIDL peaks are less with a higher pinhole
setting using PMT 3 This is in direct contrast to PMT 1,2 which
demonstrated a greater FWHM in the same reference peaks with the larger
pinholes.

Does anyone have any ideas of how opening a pinhole can increase the
resolution of a spectral system and not decrease it?    Remember there
is NO magic in confocal microscopy only the physics of light to explain
strange phenomenon.

A  PDF  is  available  on  request,  for  those who are not aware of our
November  2004  publication  in Cytometry describing the technique using
the  MIDL  lamp  “Lerner  JL Zucker, R.M.  Calibration and Validation of
spectroscopic imaging: Cytometry 62A:8-34 2004

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]