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At our Institute we also do a lot of our on the Vantage using Indo-1.
>The UV excited fluorescence signal is split by a 505nm dichroic mirror
>and the two fluorescences are collected at 395+/-25nm (Fl4) and
>525+/-25nm (Fl5). The dichroic mirror is a curious thing with a sort of
>hinge, making it infinitely adjustable. I have aligned the system with
>UV beads but get little or no signal in FL5. This may be due to the
>spectrum of the beads.
We use a different Filter combination a 530/30nm BP in the Fl-4 detector, a
405/20nm BP in the Fl-5 detector and a 475nm long pass dichroic.
I initially use Polyscience Fluorsbrite green beads which excite very nicely
with UV to check my Fl-4 detector and then use Polyscience blue beads to
check my Fl-5 detector.
I also use FCSC Indo-1 beads to check instrument once I have completed my
alignment.
If you have access to the book Flow Cytometry and Cell Sorting by Melamed et
2nd Ed. there is an excellent chapter on Calcium work and different filter
combinations.
>With Indo-1 (3uM) loaded cells, I get a nice signal in FL4, but again no
>signal in FL5.
You should get a signal in Fl-5 if all is correctly aligned if you use
green beads whcih excite in the UV and adjust the dichroic mirror.
Have you checked your Fl-5 detecter using a different probe to see if the
actuall detector is working OK or perhaps it is not sitting correctly in
it's block?
Hope this of some help.
Grace:-)
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Grace Chojnowski | |
Flow Cytometry Lab. \ /
Queensland Institute of Medical Research ____ .
The Bancroft Centre ____|---------*<<<<<<<<<<<
300 Herston Road Herston QLD 4006 - +
AUSTRALIA - +
Tel: 61-7-362 0315 - +
Fax: 61-7-362 0107 #^Q!@*X! ## + lovely!
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