SOME EXPERIENCES WITH RED-DIODE LASER (630 nm) EXCITABLE DYES ON THE FLOW CYTOMETER

We investigated the use of a cytometer equipped with a red diode laser as a possible detector for biological warfare agents (Becton-Dickinson FACSCaliburTM 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. In this mode, we wanted to differentiate between spore, vegetative bacteria, MS2 phage (virus), and ovalbumin (a toxin simulant). Our results showed that we could easily differentiate and detect the bacteria at levels of 10^3 CFU/ml with good frequency. Results with the virus and toxin were not as profound, although we were detecting the protein at 10 ng/ml about 50% of the time. Our results show that relatively simple and inexpensive cytometers with red-diode lasers as excitation sources may be an effective platform for use in a biological detector system.