Near-ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation.

Abstract

Although multiple lasers are now standard equipment on most modern flow cytometers, ultraviolet (UV) lasers (325-365 nm) remain an uncommon excitation source for cytometry. Nd:YVO4 frequency-tripled diode pumped solid-state lasers emitting at 355 nm are now the primary means of providing UV excitation on multilaser flow cytometers. Although a number of UV excited fluorochromes are available for flow cytometry, the cost of solid-state UV lasers remains prohibitively high, limiting their use to all but the most sophisticated multilaser instruments. The recent introduction of the brilliant ultraviolet (BUV) series of fluorochromes for cell surface marker detection and their importance in increasing the number of simultaneous parameters for high-dimensional analysis has increased the urgency of including UV sources in cytometer designs; however, these lasers remain expensive. Near-UV laser diodes (NUVLDs), a direct diode laser source emitting in the 370-380 nm range, have been previously validated for flow cytometric analysis of most UV-excited probes, including quantum nanocrystals, the Hoechst dyes, and 4',6-diamidino-2-phenylindole. However, they remain a little-used laser source for cytometry, despite their significantly lower cost. In this study, the ability of NUVLDs to excite the BUV dyes was assessed, along with their compatibility with simultaneous brilliant violet (BV) labeling. A NUVLD emitting at 375 nm was found to excite most of the available BUV dyes at least as well as a UV 355 nm source. This slightly longer wavelength did produce some unwanted excitation of BV dyes, but at sufficiently low levels to require minimal additional compensation. NUVLDs are compact, relatively inexpensive lasers that have higher power levels than the newest generation of small 355 nm lasers. They can, therefore, make a useful, cost-effective substitute for traditional UV lasers in multicolor analysis involving the BUV and BV dyes.