Analysis of UV-excited fluorochromes by flow cytometry using near-ultraviolet laser diodes

Authors

  • William G. Telford

    Corresponding author
    1. Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
    • National Cancer Institute, National Institutes of Health, Building 10, Room 12C121, 9000 Rockville Pike, Bethesda, MD 20892
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  • This article is a US Government work and, as such, is in the public domain in the United States of America.

Abstract

Introduction

Violet laser diodes have become common and reliable laser sources for benchtop flow cytometers. While these lasers are very useful for a variety of violet and some ultraviolet-excited fluorochromes (e.g., DAPI), they do not efficiently excite most UV-stimulated probes. In this study, the next generation of InGaN near-UV laser diodes (NUVLDs) emitting in the 370–375-nm range have been evaluated as laser sources for cuvette-based flow cytometers.

Methods

Several NUVLDs, ranging in wavelength from 370 to 374 nm and in power level from 1.5 to 10 mW, were mounted on a BD Biosciences LSR II and evaluated for their ability to excite cells labeled with the UV DNA binding dye DAPI, several UV phenotyping fluorochromes (including Alexa Fluor 350, Marina Blue, and quantum dots), and the fluorescent calcium chelator indo-1.

Results

NUVLDs at the 8–10-mW power range gave detection sensitivity levels comparable to more powerful solid-state and ion laser sources, using low-fluorescence microsphere beads as measurement standards. NUVLDs at all tested power levels allowed extremely high-resolution DAPI cell cycle analysis, and sources in the 8–10-mW power range excited Alexa Fluor 350, Marina Blue, and a variety of quantum dots at virtually the same signal-to-noise ratios as more powerful UV sources.

Conclusions

These evaluations indicate that near-UV laser diodes installed on a cuvette-based flow cytometer performed nearly as well as more powerful solid-state UV lasers on the same instrumentation, and comparably to more powerful ion lasers on a jet-in-air system, and. Despite their limited power, integration of these small and inexpensive lasers into benchtop flow cytometers should allow the use of flow cytometric applications requiring UV excitation on a wide variety of instruments. © 2004 Wiley-Liss, Inc.

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