Hello to all those people who replied to my query about 488/UV dual laser excitation on a BD FACStar Plus sorter (and to any other flowers interested in a long saga): 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
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:24 EST