Dear Doug,
You have reason to be wary, based on some of the very issues you have
brought up. However, having said that, I will tell you that RELATIVE
intensity measurements in fluorescence have a long and happy history,
starting with some of the earliest work in 1932 by Caspersson et. al. If I
were to describe the typical microspectrophotometers available from Zeiss
and Leica, you would see that there are many cross-overs to confocal:
(a) 12 bit PMTs are used as detectors
(b) an aperture is inserted to reduce the spectrophotometric effects of glare
To make results more consistent
(a) you do, indeed, need to pay careful attention to sample preparation
(b) you need to be consistent re: the size of aperture you use (i. e.,
fixed pinholes of specific diameters are much preferable to iris-type
controls)
(c) you need a broad, continuous, well-behaved standard against which to
compare your results. When I worked at Zeiss as a microspectrophotometry
specialist, we had a piece of fluorescent glass. If you call Tom Calahan
(914-681-7733), he can probably guide you to a source. When I later worked
at Reichert /Cambridge/AO (now Leica), we used a piece of fluorescent glass
which was divided into 4 quadrants. One was left bare (100% Intensity),
and each of the others was coated with varying amounts of neutral density
filter. Instead of calibrating against one intensity, we plotted 4
different values and established a contrast response function which was
then used to correct system irregularities. I suspect that a company like
Omega Optical or Chroma could reproduce a similar standard fairly easily
and you would just need to write a short piece of programming to fit in
with your control and analysis program for the system correction.
This is one of the few applications in which I would really encourage your
going to a 12 bit (4096 gray level) system rather than the typical 8 bit
systems offered routinely through standard image analysis packages.
There have been several independent inventors who have investigated the
integration of microspectrophotometry with confocal. You may want to
discuss the details further with them. The two who come to mind quickly
are Dr. Ted Dixon (now with Biophotometrics: 519-886-9013) and Coco
Montague (now with Genetic Microsystems: 781-932-9333). Please send my
regards to both if you speak with them.
Best regards,
Barbara Foster
Consortium President
Microscopy/Microscopy Education ...Educating microscopists for greater
productivity.
125 Paridon Street Suite 102 Springfield, MA 01118
PH: (413)746-6931 FX: (413)746-9311 email: [log in to unmask]
Visit our web site <http://www.MME-Microscopy.com/education>
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MME is America's first national consortium providing
customized on-site workshops in all areas of
microscopy, sample preparation, and image analysis.
At 10:05 AM 10/29/99 -0700, Doug Cromey wrote:
>Confocalists,
>
>I am interested in some feedback from list subscribers as to whether
>fluorescence intensities in confocal images can be quantitated. There seem
>to be two schools of thought about this issue (with some "gray" in
>between), one that does quantitation & wonders why I would raise this
>issue, and another that is wary of performing quantitation on confocal
>images due to the many difficult to control variables involved (optical,
>staining, and sometimes inter-operator).
>
>I would appreciate hearing under what conditions (if any) you would
>consider quantitation to be a scientifically appropriate form of analysis.
>
>I would also appreciate it if you could provide literature references that
>would help me sort out this issue.
>
>I am currently in the "wary" camp, but I'm trying to be open minded about
>this. I will post a summary of the replies to the list.
>
>Yours,
>Doug
>.....................................................................
>: Douglas W. Cromey, M.S. Dept. of Cell Biology & Anatomy :
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