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I have followed this thread with some interest. I am curious as to the current state of the art
in microbeads. For instance, can one buy and introduce into the specimen microbeads whose fluor concentration
is known a priori?
Badri Roysam
Professor, Department of Electrical, Computer and Systems Engineering
Associate Director, NSF Center for Subsurface Sensing & Imaging Systems (CenSSIS ERC)
Rensselaer Polytechnic Institute
110 8th Street, Troy, New York 12180-3590.
Office(JEC 7010): 518-276-8067, Lab(JEC 6308): 518-276-8207, Fax: 518-276-8715
Email: [log in to unmask], Web: http://www.ecse.rpi.edu/~roysam
----- Original Message -----
From: Scott Howell [mailto:[log in to unmask]]
To: [log in to unmask]
Subject: Re: Quantitative fluorescence reference needed
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> George,
>
> Thank you very much for the refs I will get after them.
>
> Scott J. Howell, Ph.D.
> Manager, Imaging Module
> Visual Sciences Research Center
> Case Western Reserve University
> Institute of Pathology Room 106
> 216-368-2300
> http://www.case.edu/med/vsrc/
>
> ----- Original Message -----
> From: George McNamara <[log in to unmask]>
> Date: Thursday, May 31, 2007 9:35 pm
> Subject: Re: Quantitative fluorescence reference needed
> To: [log in to unmask]
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >
> >
> > J Swedlow et al Measuring tubulin content in Toxoplasma gondii: a
> > comparison of laser-scanning confocal and wide-field fluorescence
> > microscopy.Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2014-9.
> > PMID: 11830634
> >
> > Jason also has the following chapter in (I believe) 2nd and 3rd
> > editions of MCB's video microscopy:
> >
> > Quantitative fluorescence microscopy and image deconvolution.
> > Methods Cell Biol. 2003;72:349-67. Review. No abstract available.
> > PMID: 14719340
> >
> > Live cell imaging using wide-field microscopy and deconvolution.
> > Cell Struct Funct. 2002 Oct;27(5):335-41. Review.
> > PMID: 12502887
> >
> >
> > Using a PMT based confocal is likely to involve a lot of
> > evaluation
> > before anyone can be confident that intensity level 2000 means
> > twice
> > as many photons as 1000. As Jim Pawley mentions in his Handbook,
> > those two numbers likely refer to 10 (plus or minus square root of
> > 10) and 5 (plus or minus square root of 5) photons. If you want to
> > use confocal, see:
> >
> >
> > <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
> db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16872427&query_hl=16
> &itool=pubmed_docsum>Cho
> > EH, Lockett SJ. Calibration and standardization of the emission
> > light path of confocal microscopes.
> > J Microsc. 2006 Jul;223(Pt 1):15-25.
> > PMID: 16872427
> >
> > I don't think they mention that the Hamamatsu R6357 PMT used in
> > the
> > Zeiss LSM510 (and many other PMTs) have a gain is proportional to
> > voltage to the seventh power (voltage is the 87 ... 1250 slider in
> > the LSM510). I found that gain = (voltage/1000)^7 emulates fig 5
> > of
> > the Hamamatsu data sheet for the R6357. See
> > http://www.datasheetcatalog.com/hamamatsucorporation/21/ for a
> > Hamamatsu data sheets.
> >
> >
> > For quantitative fluorescence, I would use a scientific grade
> > digital
> > camera in low gain mode, such as a Hamamatsu ORCA-ER (making sure
> > the
> > offset slider is set appropriately is also important ... for the
> > ORCA-ER, when in doubt, set offset to max, such as in MetaMorph's
> > Acquire dialog ... I believe the high gain is 2x the low gain for
> > the
> > ER). Once I figure out how good the deconvolution software that I
> > have access to is, I'd crunch Z-series with that (or better yet,
> > have
> > ultra flat cells). I would also adjust the arc lamp to get as
> > uniform
> > illumination, and image at least 100 cells per "treatment", being
> > careful to minimize overhead. And don't forget the controls
> > (hundred
> > of each of them, too).
> >
> > best wishes,
> >
> >
> > George
> >
> > At 09:42 AM 5/31/2007, you wrote:
> > >Search the CONFOCAL archive at
> > >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > >Dear List,
> > >
> > >
> > >I have a lab with very little microscopy experience wanting to start
> > >doing some quantitative fluorescent measurements on their
> > proteins of
> > >interest. Does anyone know of a good article (not to technical)
> > that I
> > >can have them read to begin to get them up to speed?? Just
> > looking for
> > >a decent "for beginners" article. Thanks.
> > >
> > >Scott J. Howell, Ph.D.
> > >Manager, Imaging Module
> > >Visual Sciences Research Center
> > >Case Western Reserve University
> > >Institute of Pathology Room 106
> > >216-368-2300
> > >http://www.case.edu/med/vsrc/
> >
> >
> >
> >
> >
> >
> > George McNamara, Ph.D.
> > University of Miami, Miller School of Medicine
> > Image Core
> > Miami, FL 33010
> > [log in to unmask]
> > [log in to unmask]
> > 305-443-7444x7430 (temporary - hotel room)
> > 305-443-8436 office
> > 323-251-8878 cell
> >
> >
>
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