Electronic Noise/Detection Sensitivity
Kristi R Harkins (kharkins@iastate.edu)
Wed, 03 Nov 93 18:02:28 CST
We are attempting to determine the fluorescence detection limits of our
EPICS 753. We would like to be able to detect and sort a small circular
chromosome for one project and FISH on metaphase chromosomes in
suspension for another. I have some old (4years) Flow Cytometry Standards
fluorescence equivalent units (FEU) beads, the lowest of which is 8K FEU of
fluorescein. We cannot see anything with these via flow. We can see 36K FEU
above the noise level. I am assuming that our detection level is probably
lower than this because the beads are old and have lost FEU with time. Coulter
specs state a sensitivity level of 50 molecules of fluorescein. I am using the
50 micron quartz tip with 488 at one watt and a gain of 50/high voltage of
1200. My noise level encompasses about 30 channels on log green and 19
channels on linear green and is independent of the laser power, so I therefore
conclude that it is electronic. Our cytometer is old (7 years) and I
suspect that as the parts get older they generate more noise. Would a new
PMT be worth a try? I hesitate to replace anything else because all the new
parts from Coulter are actually referbished old ones. Has anyone experience
with adding shielding and if so of what material and where should it be
located? How about replacing cables? Are there better ones out there with BNC
connections? Once again, any experiences you have had, hints, suggestions,
stories of the lowest fluorescing sample you have detected and FEU numbers
if you have them. I will summarize these responses in addition to the
chromosome sorting information that I have received.
Thanks again,
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^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Kristi R. Harkins, Lab Director <> Office: 515/294-2472
Cell & Hybridoma Facility <> Lab: 515/294-8504
Iowa State University <> FAX: 515/294-0453
1104 Molecular Biology Bldg. <> E-mail:kharkins@iastate.edu
Ames, IA 50011 <>
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EMAIL robinson@flowcyt.cyto.purdue.edu
