Some Experiences with Red-Diode Laser (630 nm) Excitable Dyes on the Flow Cytometer

摘要

We investigated the use of a cytometer equipped with a red diode laser as a possible detector for biological warfare agents (Becton-Dickinson FACSCalibur(TM) equipped with a red diode laser in addition to the Argon laser). These instruments are smaller, require less power, and also have a reduced logistics burden. We decided to focus our efforts on YOYO-3, TOTO-3, and TOPRO-3, since we have worked with the blue excitable analogs of these dyes. Our initial studies with YOYO-3 and TOTO-3 showed little, if any, staining of the bacteria. A possible explanation is that these dyes are probably too large to properly intercalate with the DNA without some prior treatment. TOPRO-3, on the other hand, is a much smaller molecule than either of the dimeric cyanine dyes that were evaluated. Consequently, we were able to obtain histograms with both vegetative and spore-forming bacteria. The differences in the histograms showed that we could readily differentiate between spore-forming and vegetative bacteria. The background signals obtained with this dye were also significantly lower than those that we observed with either the blue or green excitable dyes. We then used this dye as a detector during Joint Field Trial V.