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Dear,
I think we should be carefull to link increase of sensitivity with better
cut-off at the shorter wavelengths. Because this part of the emission
spectrum has a hughe overlap with the absorption spectrum. An overlap of
emission and excitation will increase reabsorption of the fluorescent signal
and result into loss of fluorescence at higher concentration. If
quantitative information should be obtained we even should try to eliminate
the reabsortion as much as possible.
Due to the geometry of the microscope the numerical aperture will affect
this reabsorption phenomenon, and hence interfere with the results when we
use very steep cut-off filters near the absorption maximumum of the dye.
These are statements already made long before confocal microscopy but are
valid for all fluorescence also cuvet fluorescence.
For further information you can look at:
Rigler A: Microfluorometric characterization of intracellular nucleic acids,
and nucleoporteins by acridine orange. Acta Physiologica Scandinavica Vol 67
Supplement 267. (1966)
Tanke H., Van Oostveldt P., Van Duyn P.: a parameter for the distribution of
fluorophores in cells derived from measurements of innerfilter and
reabsorption phenomenon. Cytometry 2(6) p359-369 (1982)
Van Oostveldt P Bauwens S.: Quantitative fluorescence in confocal
microscopy. The effect of the detection pinhole on the reabsorption and
innerfilter phenomena. J.Microscopy 158(2)p121-132 (1990)
Bye
Patrick
----- Original Message -----
From: "Stephen Cody" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Thursday, August 29, 2002 6:41 AM
Subject: Re: Sensitivity
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1Ļocal
>
> Dear Mark,
>
> Having had the sales talk from our Leica rep., perhaps I can explain what
they are on about.
>
> They are really comparing their result, with a "typical" setup with
dichroics.
>
> Consider exciting FITC with a "typical" dichroic system. On our confocal
we would excite with 488nm and choose the 527LP as our primary dichroic,
that's what our system is setup with. But as the peak emission of FITC is
around 520nm we are losing more than half the spectra of FITC, due to the
inflexibility of our existing filters.
>
> If we had an AOBS we would be able to set it up as a 500nm long pass
filter, or even a 490 LP. As the cutoffs are much steeper compared with
dichroics. So if I were equipped with an AOBS, I would be able to collect
the whole FITC spectra.
>
> So what I believe Leica are saying is that with dyes with a small Stokes
shift, you can collect far more of the spectra than you would with a
dichroic setup. I don't believe they are comparing the efficiencies of
transmission of the device per se, although that may form part of their
calculation.
>
> Disclaimer: I'm a poor scientist, with no financial links with Leica. In
fact I don't even have a Leica or an AOBS equipped system. Just thought I'd
translate their "sales speak" into english!
>
> Cheers
>
> Stephen H. Cody,
> Microscopy Manager,
> Ludwig Institute for Cancer Research,
> Post Office Royal Melbourne Hospital,
> Parkville, Victoria 3050, Australia.
>
> Tel: +61 3 9341 3155 (BH) +61 3 9341 3158 (@work AH)
> Fax: +61 3 9341 3104
> email: [log in to unmask]
> http://www.ludwig.edu.au/labs/confocal.html
>
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