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Some years ago Dr. Heddy Zola & I worked on optimising a Flow Cytometer
to get the detection limit down to a few tens of antigen molecules.
The work was done on a BD FACS Analyser with a mercury lamp rather than a
laser, but we were able to get similar results on a FACS IV sorter.
That meant optimising a lot of things, and some compromises; The major
limit was that to optimise a measurement, it had to be single colour!
We used PE as the brightest fluorochrome we could use in our systems.
We tried different suppliers, different batches etc. of reagents.
I think we used Biotin-Avidin, but not sure.
We optimised excitation wavelength for PE; the 546 line from Hg lamp, or
the 514 line from argon laser were the best available.
We optimised the fluorescence collection bandwidth and wavelength, looking
for the best signal to noise ratio.
We optimised the detector sensitivity, (PMT HV) again for signal to noise
ratio.
I think similar methods could now be applied to a multi-laser instrument
to get multicolour measurements, but that's a whole new ball game!
Given time I could find a reference, it was published around 1987 or 1988
I think. Can't remember which journal either!
Have fun!
Joseph
Joseph Webster (O.I.C. Flow Cytometry & Communications) ===
Centenary Institute of cancer Medicine & Cell Biology / \
Locked Bag No.6 || o o ||
Newtown, NSW 2042 || | ||
AUSTRALIA. | \___/ |
Ph: 61-2-565-6110 \ /
Fax: 61-2-565-6101 |||||
E-Mail: J.Webster@centenary.usyd.edu.au
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