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Dear John
Howard Shapiro wrote the excellent book entitled "Practical Flow
cytometry" . One of his fundamental rules in the field of flow
cytometry deals with the statement that there is NO magic. in flow
cytometry science. Now flow cytometry involves lasers, dyes, optics
and fluoresces --this sounds very very similar to confocal microscopy.
From my experience in both of these technology we observe phenomenon
that are very strange and can not be explained easily by known
principles. .
"there is no magic in science" refers to this concept of searching for
an explanation. . Now for those of us who do FRET or think about doing
FRET--this statement may make alot of sense.
Also some light scattering effects observed on a confocal microscope
have an explanation that is based in the physical principles of light
and how it interacts on various slits and optical components of the
confocal mciroscope.
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]
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| | 02/03/2005 10:15 AM |
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| Subject: Re: Pinhole and resolution |
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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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