Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Greetings,
I would recommend a dilution series of a stable fluorochrome in
chambered coverslips for "sensitivity" testing. A laser power meter,
calculator and notebook are also needed. Competing microscopes should
be set to a standardized laser power (measured carefully with a laser
power meter) and equivalant dichroic and objective. Zoom level, scan
frequency or dwell time (this is where the calculator is handy), scan
resolution, spectral detection range, detection bandwidth, averaging or
summing, and bit-depth should all be standardized. If these factors are
not standardized, then a comparison is not very quantitative. Then I
would set the PMT gain/offset voltages such that the mean pixel value of
a lambda section at the fluorochrome emission peak is about 50% of the
available range (eg. 130 on a scale of 0-255). Then acquire lambda
stacks. I would do this using successively higher dilutions until the
point at which the mean pixel value of the noise floor (lambda sections
which should have a mean intensity value of zero) reaches 50% of the
lambda section with the highest mean pixel value in the same stack.
This is easily visualized on a plot of mean intensity as a fct of
wavelength. Another method would be to acquire lambda stacks as above
and measure the CV (std. dev/mean value [Zucker and Price, Methods
18,447-458, (1999)]) of the pixel values integrated over the lambda
stack. A plot of CV vs. decreasing fluorochrome conc. should reveal a
point at which the CV decreases and flattens out. This is because the
PMT noise becomes the overriding source of signal intensity in all the
lambda sections; when this happens one no longer gets a distinguishable
emission profile on a plot of intensity vs. wavelength of acquisition.
This is a time consuming approach, but it would yield a useful
comparison of detection efficiency for the lambda detection schemes used
by various manufacturers.
-Karl
Robert Zucker wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I am glad to see that equipment manufactures are now testing their
>equipment to deliver performance specifications of spectral separation
>and sensitivity. .
>Could you explain to the confocal users on this web site how one
>measures sensitivity on a confocal system? What is your test that you
>are planning on using to show the superiority of your system and to
>demonstrate "sensitivity".
>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]
>
>
>
>|---------+------------------------------------>
>| | Will Rogers |
>| | <Will.Rogers@LEICA-MICROS|
>| | YSTEMS.COM> |
>| | Sent by: Confocal |
>| | Microscopy List |
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>| | O.EDU> |
>| | |
>| | |
>| | 10/21/2004 03:46 PM |
>| | Please respond to |
>| | Confocal Microscopy List |
>| | |
>|---------+------------------------------------>
> >-----------------------------------------------------------------------------------------------------|
> | |
> | To: [log in to unmask] |
> | cc: |
> | Subject: Re: Comparison Shopping for Confocal |
> >-----------------------------------------------------------------------------------------------------|
>
>
>
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>This reply is from Leica Microsystems
>
>Mike,
>
>Leica confocal microscopes can optically separate physiological levels
>of CFP / YFP or for that matter any XFP combination.
>
>We would welcome a public comparison of confocal systems on a common
>sample set. Leica will be on display at the American Society for
>Neurosciences meeting in San Diego next week and would be happy to
>participate in a comparison of system sensitivity and spectral
>separation.
>
>Will Rogers
>Vice President
>Leica Microsystems Inc.
>610-321-0460
>[log in to unmask]
>
>
>
>
> mancini <[log in to unmask]>
> Sent by: Confocal Microscopy List To:
> <[log in to unmask]> [log in to unmask]
> DU
> cc:
> 10/21/2004 12:28 PM Subject:
> Please respond to Confocal Microscopy Comparison Shopping for
> List Confocal
>
>
>
>
>
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>
>
>>Alby,
>>
>>When you say you especially like how the Leica works well with low
>>signal strength, is this something you directly compared against other
>>systems? In our work, we continue to be reminded that only the very
>>lowest levels of XFP expression are "relevant." Is this where you say
>>the
>>Leica still can perform well? If "unmixing" is used to clean up
>>noisy/loud autoflourescence, that's one thing, but trying to unmix
>>XFP's at
>>~physiological levels is not something we've had any success with,
>>unfortunately. I'd
>>love to see a direct comparison between spectral scopes with such an
>>approach.
>>
>>Cheers,
>>Mike
>>
>>*****************************************************
>>Michael A. Mancini, Ph.D.
>>Associate Professor
>>Director, Integrated Microscopy Core
>>Department of Molecular and Cellular Biology
>>M803 DeBakey Building
>>Baylor College of Medicine
>>Houston, TX 77030
>>713 798 8952
>>713 798 3175 fax
>>713 408 0179 cell
>>[log in to unmask]
>>http://www.bcm.tmc.edu/mcb/faculty/mancini.html
>>http://microscopy.bcm.tmc.edu
>>
>>On Oct 20, 2004, at 11:11 PM, Alberto Diaspro wrote:
>>
>>
>>
>>
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>>Dear Tony,
>>>
>>>For us the choice would be (in order)
>>>Leica TCSP AOBS SP2
>>>New Nikon C1
>>>Olympus FV1000
>>>Leica and Nikon are the ones we work with in our lab, and we made our
>>>
>>>choice after some comparative tests. I think Leica is a different
>>>class
>>>
>>>with respect to Nikon and Olympus. Moreover, the one I trust,
>>>following
>>>
>>>the proper calibration as for all the microscopes, in terms of
>>>specttral detection is Leica. This system works very well in our lab,
>>>
>>>also coupled, by home made set-up, to a Chameleon Ti-Sapphire for
>>>
>>>
>TPE
>
>
>>>ps/fs, serving several projects involving protein trafficking. The
>>>Nikon C1 works well, too. Unfortunately the current version we have
>>>does not have improved spectral abilities. What we are impressed
>>>
>>>
>about
>
>
>>>the Leica is the good spectral separation even when you have a poor
>>>signal to noise ratio. As last remark, we found our system works well
>>>
>>>also when you have an UNKNOWN spectrum. This can happen also when
>>>considering changes in spectral proprties of fluorescent molecules.
>>>
>>>
>In
>
>
>>>many cases it seems high risk to select a fluorescent molecule
>>>fingerprint to track it.
>>>All my best
>>>Alby
>>>
>>>
>>>Il giorno 20/ott/04, alle 21:46, Tony Eissa, MD ha scritto:
>>>
>>>
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>> Dear List
>>> We have $ 500,000 that is burning a hole in our pocket for a
>>>confocal.
>>>
>>>
>>>
>>> I would appreciate any input comparing Zeiss LCM510 meta,
>>>Olympus
>>> FV1000, and Leica. The microsocpe will be shared by several
>>> investigators working mostly on protein trafficking in cell
>>>culture
>>> models. Thanks.
>>>
>>> --
>>>
>>> N. Tony Eissa, MD
>>> Baylor College of Medicine
>>> One Baylor Plaza- BCM285 Rm 672E
>>> Houston, TX 77030
>>>
>>> Tel (713) 798-3360
>>> Fax (713) 798-5780
>>> e-mail: [log in to unmask]
>>>
>>>
>>>
>>>
>>>
>----------------------------------------------------------------------
>
>
>>>--
>>>
>>>---------------------------
>>>Alberto Diaspro, Department of Physics, University of Genoa
>>>Via Dodecaneso 33, 16146 Genova, Italy
>>>facsimile +39-010314218 - voice +39-0103536426/480/309
>>>URL: http://www.lambs.it
>>>http://www.wiley.com/WileyCDA/WileyTitle/productCd-0471409200.html
>>>
>>>
>>>
>----------------------------------------------------------------------
>
>
>>>--
>>>
>>>--------------------------
>>>
>>>
>>>
>>>
>
>
>
>______________________________________________________________________
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>
>
--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu
|
