Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis

Authors

  • Richard M. P. Doornbos,

    1. Applied Optics Group, Faculty of Applied Physics, University of Twente, Enschede, The Netherlands
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  • Bart G. de Grooth,

    Corresponding author
    1. Applied Optics Group, Faculty of Applied Physics, University of Twente, Enschede, The Netherlands
    • Applied Optics Group, Faculty of Applied Physics, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
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  • Yvonne M. Kraan,

    1. Applied Optics Group, Faculty of Applied Physics, University of Twente, Enschede, The Netherlands
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  • Carel J. van der Poel,

    1. Applied Optics Group, Faculty of Applied Physics, University of Twente, Enschede, The Netherlands
    Current affiliation:
    1. Philips Optoelectronics Centre, Philips Research Labs, P.O. Box 80000, 5600 JA Eindhoven, The Netherlands
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  • Jan Greve

    1. Applied Optics Group, Faculty of Applied Physics, University of Twente, Enschede, The Netherlands
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Abstract

This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm. diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently 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 sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime. © 1994 Wiley-Liss, Inc.

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