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FACStar Plus with active 7th detector option (to enable 5 colours)
three lasers: I-70 running 488 at 200 mw, I-90 running UV at 35 mw, SP HeNe
at 35 mw. I-70 is laser one. UV and HeNe are collinear, at ~20 microsecond
delay.
Iris reduced to minimum possible (about half closed) in FL4 channel.
Pinhole in place in front of APC PMT.
Filters: 640 LP dichroic, 670/20 BP and a GG420LP (absorption filter) in
front of APC PMT and a DF424 in front of AMCA PMT.
Here comes the puzzle:
I did not think that APC could be excited by UV. But we see a signal in the
APC channel from FCSC APC beads when only the UV laser is unblocked. It is
10% the intensity seen from the same beads when only the HeNe laser is
unblocked. (In both cases, the 488 beam is unblocked.) I thought that we
were seeing light scatter from the UV laser that was penetrating the 640
and the 670, so that is why the 420 is there as well. Use of the 420 made
no difference other than reducing all signals by about 20%. So now I seem
to be seeing true red fluorescence of APC excited by UV illumination. My
paradigm shifts! My flow cytometric bubble bursts! (I don't know so much
after all!!!)
Seriously, does anyone have any ideas as to whether APC really could be
doing this. Molecular Probes can't tell me for sure. (They are "too short
on resources" to do a spectrofluorometer run to check) Or more likely,
what am I missing here in instrument setup?
Thanks very much.
_______________________________________
Dave Ehman
Technical Applications Guy
BD Canada
PH: (800)267-5586 Xt. 2004
Fax: (613)836-3580
Email: dehman@hookup.net
_______________________________________
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