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.