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I quite agree with Bob's analysis, and would just add the following
two points.
1. With a 5nm slit width (the minimum on the Leica), to sample at
Nyquist one should select 2nm steps - one should then get 5nm
resolution and as Bob says, that is achievable at the short end
of the spectrum. It's not necessarily all that time-consuming
since there's no need to stick with 512 * 512 frame size. But
it is essential to select 12 bit sampling.
2. The problem with a broad field light source is that it is
not being imaged confocally so the path of the rays arriving
at the detector may not be what the system designers intended.
It is clearly stated by Leica that opening the pinhole will
worsen the spectral resolution. Even with the pinhole closed,
one is not imaging the source confocally since it is not a focussed
spot at the sample plane, so the effect may be similar. That is
probably why Leica don't like this test. I don't see the problem
in using the laser lines - one can use a power meter if one wants
to reproducibly check sensitivity as well as wavelength accuracy.
Guy
> 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
> <[log in to unmask] To:
> [log in to unmask]
> ET> cc:
> Sent by: Confocal Subject: Re: Pinhole and
> resolution
> Microscopy List
> <[log in to unmask]
> UFFALO.EDU>
>
>
> 02/03/2005 05:26 PM
> Please respond to
> Confocal Microscopy
> List
>
>
>
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Robert,
>
> In your original query regarding this thread, you stated, "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." This is in reference to the MIDL spectral pattern.
>
> Unless you have already ruled this out, could this not be at least in
> part the explanation? Each PMT in the system, I assume is part of a
> different light path (including optical components), and opening the
> pinhole to a much larger area than a single Airy disk could affect
> the optical transfer function in different ways for different
> wavelengths. In PMTs 1 and 2 opening the pinhole reduces resolution
> and accuracy (I am not exactly clear what you mean by accuracy in
> this case; relative peak height, wavelength estimate, but you get
> peak broadening).
>
> Although PMT 2 and PMT 3 are the same Hamamatsu tube, the light path
> to each is different, correct? And you also mention that they operate
> at different voltages (lower for PMT 3). It has been my experience
> with PMTs that there can be significant differences from one tube to
> the next even when nominally identical according to the manufacturer.
> Their "best" or optimal operating voltages can be hundreds of volts
> apart. The fact that you are using a lower voltage with PMT 3 might
> be because of tube differences.
>
> On the other hand, it would be expected that PMT 3 using a lower
> voltage means that you are less likely to be close to tube saturation
> compared to PMT 2. Opening up the pinhole might provide the extra
> sample signal, as Anda suggested, but the lower voltage might still
> keep you in a linear gain region of the PMT, whereas flattening
> (saturation) of the PMT 1 and 2 signals might start appearing if the
> tubes are operating closer to the non-linear gain region of the PMTs
> and the pinholes are greatly increased and the tubes are over
> illuminated.
>
> One indication of this possibility is if the peaks are actually close
> to their expected positions but their FWHMs are broadened. Another
> test would be to use neutral density filters or equivalents to
> decrease the source brightness and determine if the FWHM changes with
> the brightness of the source, as well as, their apparent peak
> wavelengths.
>
> Cheers,
>
> Mario
>
>
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>HI Anda
>>The PMT 3 actually operatives at less voltage than PMT 2 at an airy
> disk
>>of 1 --both are the same type of Hammatsue PMTs.
>>That may imply that there is some extra scattered light that is
>>entering this PMT at this voltage.
>>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]
>>
>>
>>|---------+------------------------------->
>>| | Anda Cornea |
>>| | <[log in to unmask]> |
>>| | Sent by: Confocal |
>>| | Microscopy List |
>>| | <[log in to unmask]
>>| | UFFALO.EDU> |
>>| | |
>>| | |
>>| | 02/02/2005 06:22 PM |
>>| | Please respond to |
>>| | Confocal Microscopy |
>>| | List |
>>| | |
>>|---------+------------------------------->
>>
>>>-------------------------------------------------------------------------------------------------------------|
>
>> |
>>|
>> | To: [log in to unmask]
>>|
>> | cc:
>>|
>> | Subject: Re: Pinhole and resolution
>>|
>>
>>>-------------------------------------------------------------------------------------------------------------|
>
>>
>>
>>
>>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Hi Robert!
>>
>>How is your signal to noise in PMT-3? If poor, it would certainly
>>benefit from increasing the pinhole and result in a better perceived
>>resolution.
>>
>>Anda
>>
>>
>>
>>Anda Cornea Ph.D.
>>Head of the Imaging and Morphology Core
>>Oregon National Primate Research Center
>>Oregon Health & Science University
>>(503) 690-5293
>>
>>>>> [log in to unmask] 02/02/05 9:44 AM >>>
>>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]
>
>
> --
> ________________________________________________________________________________
>
> 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]
>
--
Associate Professor Guy Cox
Electron Microscope Unit,
University of Sydney,
NSW 2006, Australia
Phone:+61 2 9351 3176 Fax:+61 2 9351 7682
http://www.guycox.net
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