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Date: 02 Feb 94 07:59:01 EST
From: Marc.Langweiler@Dartmouth.EDU (Marc Langweiler)
Subject: Sci Fi (ve) mystery
To: cytometry@flowcyt.cyto.purdue.edu
It came to pass that Curley found himself in northern New England, trying
to do 3-color surface marker phenotyping. It so happens that he was using a
cytometer from a company that Moe had gone to work for, based in south
Florida. Amazingly, when he went looking for third color conjugates, he
winds up talking to Larry, who had gone to work for a company in northern
California specializing in PE-Cy5 conjugated antibodies!
Curley bought several conjugates, and found that some of them, including
CD3 and CD45 were bright as can be. He couldn't figure out why conjugates of
CD33, CD10 and CD19 were so dim. He called Larry, who told him that his
problem was probably due to a bum PMT in FL3, after making sure that he was
using an appropriate filter, a 645 LPA. In fact, said Larry, I know of
several other instances where users had to struggle with their cytometer
manufacturers until they got a good PMT. One guy had to try 5 of them before
he found one that worked!
So Curley calls Moe, and tells him this, and Moe says bull#$%$! The
problem is the conjugate. It's going to be dimmer than what you're used to
seeing. For chrissakes, just look in their catalog...You excite the PE and
it emits at a peak of 575. You now ask it to excite the Cy5, which is
excited at 625-650. There ain't no way that you going to hit Cy5 with
anything that's going to excite it much. So, bag that junk and use something
that we make.
By now Curley's really flustered. You know, starts that banging himself on
the side of his head. But, since he's gotten a little smarter since his last
gig, and has discovered cyberspace, he decides to ask the rest of the
cytometry community for help.
........
The short answer is: These fluorochrome conjugates work great with 488nm
excitation. (See below for details.) Detectors and filters are likely
candidates. Moe should go back to throwing cream pies....
The long pass filter sounds fine, but what are the other dichroics? Also, the
PMT could affect detection. Either it's bad, but more likely, is the location
and/or alignment. That is, if it sits third in line after a series of dichroics
each of which picks out a waveband, the light intensity drops by the time you
get to PMT3. (For example, if each filter is 90% efficient in passing red light
and there are 4 filters then, you have only about 66% of the light after the
last filter.) Alignment of the PMT to properly illuminate the photocathode
could also be a problem.
Flurochrome conjugates work by resonance transfer not by fluorescence emission
and re-absorption; PE-Cy5 is a stellar synthetic example. For energy transfer
to work, you need a pair of fluorochromes that have absorbance and emission
spectra that overlap, are located about 10A apart, and have the "proper"
spatial orientation. Light energy absorbed by the short wavelength absorbing
fluorochrome is not emitted as fluorescence, rather it can be transferred
directly to the longer wavelength absorbing fluorochrome. (With PE-Cy5
conjugates there is some fluorescence emission of PE--coupling is not perfect.
Carl Stewart has shown a nice overview of this.) PE-Cy5 pair is very
effectively excited by 488nm light with good emission around 670nm. It works
very well with air-cooled argon lasers. Conjugates are available from (by
alphabetical order) Amac, CalTag, GenTrak, Gibco, and Pharmingen (among
others).
This coupling is a good example of how photosynthesis works. Chlorophyll
absorbs in the blue and red ends of the spectrum; light in the green to near
red parts of the spectrum is used very inefficiently. Accessory pigments allow
the absorption of light in this region, and thus a selective advantage for
those organisms that contain them. Accessory pigments in cyanobacteria, red
algae, and cryptomonads include the phycobiliproteins: phycocyanin (PC) and
allophycocyanin (APC); many also include phycoerythrin (PE).
Arranged in order of absorption, they are PE-->PC-->APC-->chlorophyll. (PE can
transfer directly to APC as well.) These fluorochromes allow the organism that
contains them to harvest light in the green to red parts of the spectrum and
transfer it directly to chlorophyll. In seawater, the deepest growing algae
are the red algae since they are able to use green light which is the
predominant wavelength at depths (blue light is scattered by particulates, red
light is absorbed by water).
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