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Hi Bob,

I completely agree with you. The manufacturers should be held
accountable, especially since you demonstrated that with proper
servicing, each PMT, and its pathway, can be adjusted to yielded the
same result.

I am wondering if the service adjustments made that you refer to,
which resulted in consistent spectral results from each PMT, happen
to involve any pinhole and PMT voltage re-settings that were recorded
and could be referred to in the current context? I also wonder how
the spectral quality of each PMT of the current circumstance matches
the spectra obtained post servicing that was referred to in the paper?

For example, with a pinhole of 1 Airy diameter are the spectra of
PMTs 1, 2 , and 3 superimposable on the data obtained previously for
tubes 1 and 2 but not 3, since the latter has apparent broadening but
not 1 and 2? What about S/N?

Forgive me, I don't have the paper in front of me, but when you
compared the three different Leicas after servicing, did PMTs 1, 2,
and 3 for each microscope yield the same spectra. I.E., was
instrument to instrument consistent as opposed to self consistency of
PMTs within a single instrument? This relates somewhat to the
following.

I am also wondering that when you originally obtained self consistent
spectra, these spectra gave you the narrowest peaks (least line
broadening) and best S/N per peak. I just want to be clear that
getting self consistent results doesn't mean that you end up with
some good but not absolutely the best spectra obtainable. It would
not be surprising that one PMT option would give you a better
spectrum particularly over a certain wavelength range. In your SP1,
it was mentioned that PMT 2 and 3 use the same PMTs so presumably
they would have the same quantum detection efficiency (QDE). PMT 1
assuming it is a different PMT would have a different QDE. I admit to
being ignorant when it comes to the SP1 but to get spectra from
different PMTs to overlay means that they have to be calibrated and a
correction curve (or function) has to be estimated to account for the
different light sensitivities at different wavelengths. As I have
indicated before, PMTs can be different even if they nominally have
the identical part number. They can also change with time and with
the exact area of exposure on the photo-cathode. Again, the service
records should be able to tell you if the QDE corrections were
re-estimated, with new parameters determined so that "computed"
outputs match for each PMT. The latter is made mostly irrelevant if
some kind of detector ratio system is used but I assume that it is
not part of the standard MIDL assembly, that was put together for
instrument comparison. (Bob, I apologize if its in the paper).

As far as the slider, it would seem that significant differences
should show up in S/N, and as pointed out, that would give better
spectra if the 1 Airy setting is providing too few photons. Of
course, using a 10x lens with a lamp should provide a lot of photons.
Which brings up the light PMT saturation issue.

Good luck, Bob. I look forward to your solution.

Mario
PS wish I could be there. I really curious. Must find your paper, it
is here somewhere




>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi Mario,
>My dilemma is that we have observed, and published, data run on three
>different Leica SP systems.  Each Leica instrument contained three PMTs
>and each reported a different spectrum from the same light source (MIDL)
>with varying accuracy, contrast and spectral resolution.  However, after
>servicing, all three PMTS in each instrument reported EXACTLY the same
>spectra with a near perfect overlay.  (See before and after examples in
>Figure 4, 9, 10, in Cytometry Vol 62A, No 1, pages 8-34, November 2004).
>This consistency suggests that all PMTs in the instrument can be at the
>same "focus".  We expect no less of our microscope objectives.
>
>I do not believe that it is too much to ask for the output of each of
>the PMTs in our system to present consistent data especially when we
>know good consistency is possible. If these expectations are too much to
>ask, then at least the manufacturer should indicate the tolerance on
>inconsistency.  This way we can determine what is "normal" and when the
>instrument is out of specification; and we should call a technician.
>
>To respond to the idea of scanning over the laser lines.  This is a good
>first pass evaluation and demonstrates a number of useful factors. Laser
>scans demonstrate that the 5 nm step size is a wavelength sampling
>increment (WSI) that is only peripherally related to "resolution".  In
>our SP1 system the FWHM of the 488 nm line is about 5 nm; the 568 nm
>line FWHM = 8 nm; and the 647 nm line the FWHM = 13 nm.  This is
>expected because a prism produces non-linear wavelength dispersion
>(actually a 5th order polynomial fit).  Scanning in 1 nm increments (WSI
>= 1), or indeed less than 1 nm, does not change spectral resolution
>because the FWHM is determined by the optics of the instrument not the
>step size (again assuming the WSI is less than 5 nm).  On the other hand
>the experiment is very time consuming.  The MIDL lamp provides 5 major
>lines simultaneously, between 405 and 650 nm - a good accuracy check for
>a non-linear system where the greatest errors are likely to be at the
>extremes of the wavelength range.
>
>For those concerned with unmixing fluorophores, where most algorithms
>depend on good contrast and low stray-light, the MIDL lamp is "must
>have". This is best demonstrated between 500 and 650 nm where the
>spectral resolution of a prism based system is most compromised.  The
>MIDL lamp emits two major lines at 546 and 612 nm, and a prominent, but
>minor, line at 586 nm.  An acquired spectrum of the lamp enables us to
>assess wavelength ratios between the three lines and the influence that
>the two strong lines have on the 586 nm feature. Better performing Leica
>SP system are able to resolve these lines close to baseline to baseline;
>poorly performing (low contrast) instruments do not.  We have observed
>that consistent contrast and wavelength ratios cannot ever be taken for
>granted on any of the systems we have encountered.  As a bonus these
>scans can be acquired in 5 nm steps quite rapidly.
>
>While it is hard to believe that the slit-slider associated with our PMT
>3 could become "defocused", the evidence suggests that this is the case
>- just as Csucs Gabor suggested in a earlier response.  I will keep you
>informed of what happens"
>
>Best wishes
>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]
>
>
_______________________________________________________________________________

--
________________________________________________________________________________
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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