VISIBLE DIODE-LASERS CAN BE USED FOR FLOW CYTOMETRIC IMMUNOFLUORESCENCE AND DNA ANALYSIS

This report describes a feasibility study concerning the use of a visi ble diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluoresc ence measurements using the fluorophore allophycocyanin (APC). We have measured CDS positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negati ve cells and the dim and the bright fluorescent subpopulations. As a s econd fluorescence application, we chose DNA analysis with the recentl y developed DNA/RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is suffici ent to do single color immunofluorescence measurements with allophycoc yanin and DNA analysis with TO-PRO-3 The major advantages of using a d iode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime. (C) 1994 Wiley-Liss, Inc.