This harks back to a discussion in February and, while triggered again
from a consideration of the DiVa, this note is not really instrument
specific. Hoping to investigate the effect of integrating a detected
flow cytometric pulse just between the threshold breaks (without
threshold extension), I set up a spreadsheet to model the detector
pulses arising from cells of various sizes, surface or intracellularly
stained and illuminated with different laser beam diameters. This
indeed shows we need to be careful to integrate enough of the pulse but
I was surprised by something else:
The *peak* measurement seems to be very sensitive to the staining
pattern.
e.g., even for 10 micron cells and a 30 micron beam height, uniformly
intracellularly stained cells have nearly 3% higher peak than
uniformly surface stained cells (same total fluorescence). This goes
against my belief that if the beam is much wider than the cell, peak
measurement is as good as area. If the calculations are correct, they
further imply that cell size will strongly affect apparent total
fluorescence. I may have made a mistake. I invite those interested
to look at the spreadsheet at
http://www.wehi.edu.au/cytometry/PulseShapes/ and comment (I don't
mind public lambasting if it leads to enlightenment).
Frank Battye
| | < battye@wehi.edu.au
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