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I received a large range of very useful suggestions to our problem --- for
which I am very grateful.
To sum up, the way we discovered our problem was by using Spherotech rainbow
beads to try to align a new UV tube. The bead peaks (with UV excitation; 485
emission) were severely masked by a high level of noise; the noise disappeared
when we lowered the 488 laser output (revealing five bead peaks with pristine
CVs). So we were sure that the UV laser was well aligned but that noise from
the 488 laser was coming through at the "time" and "place" that should have
been reserved for the UV-excited signal. When looking at indo-1 changes using
the 485/405 filter set, the amount of 488 laser scatter coming through the 485
filter was high enough that it significantly (but not by any means completely)
damped down the magnitude of the ratio change that occurred upon calcium flux.
Of about 10 replies to my specific questions, two other labs were seeing a
485 signal that, like ours, decreased severely when the 488 laser was turned
down. Several of the other replies came from labs using 530 filters in the
FL4 channel, so wouldn't have been able to detect contaminating 488 scatter.
And one person was using 488 excitation at 60mWatts --- a level significantly
lower than the usual 100-200 mWatts so that contaminating scatter might not
have been detected here either. Therefore, I can't really comment on the
frequency of this problem --- other than to say that we weren't the only ones
with trouble! Seven of the 10 replies definitely noticed blue 488 reflections
bouncing around near the UV laser head and UV beam-steering prisms. Most
people had the two lasers separated by about 0.17 to 0.25 mm at the stream
intersection (range from 0.13 to 0.51). From this small sample, I couldn't
detect any correlation of our described problem with 488 back scatter to the
UV laser head nor with short beam separation distances.
Everyone suggested that alignment was the likely cause of our problem --- but
we were pretty much convinced that we had the lasers aligned well (and this
turned out to be the case). By steering the UV and VIS lasers slightly
differently, we were able to cut out the reflections of the 488 laser
backwards into the UV light path --- but this had no effect on solving our
noise problem. Increasing the distance between the vis and UV beams was
difficult to do --- they seemed to settle into sweet spots --- but increasing
the distance as much as we could didn't help. Nothing about changing the dead
time and/or 2nd laser window helped --- as the 488 noise was all over and not
easily avoided by these adjustments. The half mirror was also not the problem.
Is the suspense building?? In the end we solved the problem by following a
suggestion that came both from a helpful engineer at BD and also from a couple
of canny flowers. The iris that sits just before the FL3 and FL4 detectors
turned out to be both wide open and also slightly off center. When we centered
the iris carefully and then closed it down as much as possible, we were able
to decrease the 488 scatter from the stream without decreasing the UV-excited
signal (because the latter was more centered on the channel axis than was the
noise). Centering and then closing that iris has given us quite lovely peaks
from the Spherotech Rainbow beads at 485nm as well as at the usual 488-excited
wavelengths and also at 405 (excited by UV)..
My general feeling about this problem is that it explains to me for the first
time why June and Rabinowitch say that they get better calcium flux ratio
changes when using a 530/405 ratio rather than a 485/405 ratio. It also might
explain why they say that BD instruments show a smaller change in the ratio
upon cell activation than do Coulter (stream in cuvette) instruments.
Contamination of the 485 channel with scatter of the 488 excitation beam off
the stream would "dampen" any ratio changes. It's certainly possible that,
without due care, many instruments are collecting 488 scatter off the stream
into supposed UV channels. You can solve the problem by very careful,
critical tuning ---- or, alternatively, can ignore the problem by shifting the
indo ratio to 510/405 or 530/405 instead of the peak wavelengths of 485/405.
Again, thanks for all your help!
Alice
Alice L. Givan
Englert Cell Analysis Laboratory
Dartmouth Medical School
Lebanon, New Hampshire
NH 03756 USA
tel 603-650-7661
fax 603-650-6130
e-mail givan@dartmouth.edu
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web