Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi again,
I agree that confocal microscopies and all the microscopies are magical
and make us look like magicians. In that respect, it is magic.
I haven't had time to give it a lot of thought, but I think Bob Zucker's
problem may be due to z-axis misalignment of his 3rd detector. If the
pinhole were too high or too low in the z-direction, he would actually
be imaging an out of focus plane. Opening the pinhole would then allow
his in-focus specimen to be better resolved.
This is like "Car Talk" on American public radio where listeners call in
for a diagnosis of their car problems. I hope Bob tells us know what the
problem really was once he figures it out.
John
Mario wrote:
> Search the CONFOCAL archive at
> 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
>
> [log in to unmask]
> [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]
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