Deep ultraviolet 266 nm laser excitation for flow cytometry

Abstract

High dimensional flow cytometry relies on multiple laser sources to excite the wide variety of fluorochromes now available for immunophenotyping. Ultraviolet lasers (usually solid state 355 nm) are a critical part of this as they excite the BD Horizon™ Brilliant Ultraviolet (BUV) series of polymer fluorochromes. The BUV dyes have increased the number of simultaneous fluorochromes available for practical high-dimensional analysis to greater than 40 for spectral cytometry. Immunologists are now seeking to increase this number, requiring both novel fluorochromes and additional laser wavelengths. A laser in the deep ultraviolet (DUV) range (from ca. 260 to 320 nm) has been proposed as an additional excitation source, driven by the on-going development of additional polymer dyes with DUV excitation. DUV lasers emitting at 280 and 320 nm have been previously validated for flow cytometry but have encountered practical difficulties both in probe excitation behavior and in availability. In this article, we validate an even shorter DUV 266 nm laser source for flow cytometry. This DUV laser provided minimal excitation of the BUV dyes (a desirable characteristic for high-dimensional analysis) while demonstrating excellent excitation of quantum nanoparticles (Qdots) serving as surrogate fluorochromes for as yet undeveloped DUV excited dyes. DUV 266 nm excitation may therefore be a viable candidate for expanding high-dimensional flow cytometry into the DUV range and providing an additional incidental excitation wavelength for spectral cytometry. Excitation in a spectral region with strong absorption by nucleic acids and proteins (260–280 nm) did result in strong autofluorescence requiring care in fluorochrome selection. DUV excitation of endogenous molecules may nevertheless have additional utility for label-free analysis applications.