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Dear Bob,

I don't think you need to admonish this group that "Remember there is NO
magic in confocal microscopy only the physics of light".

Best wishes,

John

Robert Zucker wrote:

>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]
>

--
John J. Lemasters, MD, PhD
Professor of Cell & Developmental Biology and Surgery, and
Director of Cell and Molecular Imaging
Department of Cell and Developmental Biology
University of North Carolina at Chapel Hill
CB# 7090, 236 Taylor Hall
Chapel Hill, NC 27599-7090 USA
Tel: 919-966-5507
FAX: 919-966-7197
E-mail: [log in to unmask]