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After reading Michael's description below and on his webpage, I'm
wondering what the list can recommend as a good standard for
quantitative fluorescence.  I'm fairly new to this, but am very
interested in logging an increase in intensity of our fluorophore
(ex/em in DAPI region) in cells over time.  We don't have a confocal
setup (sorry to be slightly off-topic), and are simply using a
Hg-lamp with cooled CCD on our Nikon scope to excite the dye.  I had
considered using Molecular Probes' fluorescent beads as a sort of
standard, or possibly the MME slides described below, but these were
both just initial thoughts without real consultation into the
literature.  Can anyone provide any better insight?

Thanks much,

Dan Harrington


>A few years ago I picked up a scrap of green fluorescent plexiglass for use
>with our microscopes.  It has proved extremely useful for checking how flat
>illumination is, for putting on edge to illustrate N.A. as an angle, and as
>a rough standard.
>
>N.b. the word "rough".  For short exposures at low intensity, it works
>practically as a standard.  But early on we noted that it could be quenched
>and that IR radiation could change the properties of the fluorescence to
>get brighter; perhaps the femptosecond pulsed laser roasted the plastic.
>
>We purchased the Fluor-ref slides from www.MicroscopyEducation.com to try
>them.  They do prove useful, but because of the caveats above, they are not
>robust standards.
>It could be argued that we beat them hard; examples of the problem can be
>found at
>http://www.aecom.yu.edu/aif/instructions/fluor-ref-slides/01.htm

--
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Daniel Harrington
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Northwestern University
Department of Materials Science and Engineering
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