 |
 |
 |
 |
Hi Geoffrey,
I'm just started with analysis of sperm (on Epics Elite) and I
have the same problems like you. I think the message from 20 Jun
is only part of your discussion and perhaps you know
modifications mentioned below.
Sperms are very specific cells for FCM analysis due to their flat
shape ab. 8-10 x 4-5 x 1 microns. Such shape gives a "lens
effect" after staining with, for example, Hoechst (what about
other stains?) - fluorescence from the edge is much stronger than
from the face. Consequently, I think, we should place all
spermatozoa in the same plane. Basing on Bernoulli relationship
we can grind off the injection nozzle to the chisel shape (idea
by LA Johnson - Beltsville, Maryland and DW Dresser, UK). Such
nozzle gives a ribbon like core stream. In the ideal case all
sperms should be in one plane, but - at least in my experience -
it doesn't work perfectly. I tried nozzles with 14 and 20d.
angles with restrained results. There is the other major
modification - replacing one of PMTs instead of FS detector (idea
by mentioned authors).
It's serious modification for me - so any precise scheme would be
appreciated...
In this way we can obtain fluorescence from 0FL and 90FL
detectors and therefore distinguish different orientation of
cells. Oblique orientation should give the highest peak,
edge-to-0FL and edge-to-90FL lower peaks.
What do you think about it?
Have anyone any suggestions?
Michal
===========================================================================
Michal Bochenek
National Research Institute of Animal Production
Department of Animal Reproduction
32-083 Balice/Krakow, Poland
phone: 48 012 113211 ext.308; fax: 48 012 110294;
E-mail: mbochen@izoo.krakow.pl
===========================================================================
|
|
|
|