DPSS yellow‐green 561‐nm lasers for improved fluorochrome detection by flow cytometry

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Abstract
Introduction Blue‐green 488‐nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less‐than‐optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green helium–neons and, more recently, diode‐pumped solid state (DPSS) 532‐nm sources have previously been employed to overcome these difficulties and improve overall sensitivity for PE. In this study, we evaluate an even longer wavelength DPSS 561‐nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity. Methods A DPSS 561‐nm laser emitting at 10 mW was mounted onto a BD LSR II. Mouse thymoma cells labeled with cell surface marker antibodies conjugated to the R‐ and B‐forms of PE were analyzed and compared with conventional 488‐nm excitation using the same bandpass filters and signal travel distances. A similar analysis was carried out with cell lines expressing the red fluorescent protein DsRed, several green‐yellow excited low molecular weight fluorochromes, and a rhodamine‐based caspase substrate. Additionally, cells labeled with PE and co‐labeled with fluorescein or simultaneously expressing green fluorescent protein (GFP) were analyzed to determine if PE excitation at 561 nm with simultaneous fluorescein/GFP detection was feasible. Results The DPSS 561‐nm laser gave a several‐fold improvement in the fluorochrome to autofluorescence ratios between PE‐labeled cells and unlabeled controls. Analysis of cells expressing the fluorescent protein DsRed with the DPSS 561‐nm source gave a 6–7‐fold improvement in sensitivity over 488‐nm excitation, and gave excellent excitation of yellow‐green excited fluorochromes and rhodamine‐based physiological probes. Yellow‐green laser light also caused virtually no impingement on the spatially separated fluorescein/GFP detector, a significant problem with green laser sources, and also allowed simultaneous analysis of GFP and PE with virtually no signal overlap or requirement for color compensation. Conclusions DPSS 561‐nm laser excitation gave significantly improved sensitivity for both PE‐labeled and DsRed expressing cells, with little contamination of a typical fluorescein/GFP detector. Published 2005 Wiley‐Liss, Inc.