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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
John,
Who is to say there is no magic in the physics of light? In the x-y
plane the PSF is described by infinities and somewhat similarly in
the z-axis, just different ones. And to see the result of these
infinities (FWHM) for the different cases one must change the
relationships of physical constructs (pinholes).
So the rules say that in making big changes in the pinholes and using
different PMTs, you excite different areas of the PMT that are
perhaps running at different temperatures affecting the metal work
function interaction and you sample a very different volume (x, y,
z)s in the source. Could be the temperature and the EMF gradients in
the different volumes being compared as well.
Just a couple of thoughts.
Mario
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>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]
--
________________________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies LLC
President and CTO
ph (510) 528-2400
FAX (510) 528-8076
1561 Posen Ave
Berkeley, CA
94706
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