Analysis of violet-excited fluorochromes by flow cytometry using a violet laser diode

Abstract

Background: Low power violet laser diodes (VLDs) have been evaluated as potential replacements for water-cooled argon-ion and krypton-ion ultraviolet and violet lasers for DNA content analysis using the Hoechst dyes and 4,6-diamidino-2-phenylindole (Shapiro HMN, Perlmutter NG: Cytometry 44:133-136, 2001). In this study, we used a VLD to excite a variety of violet-excited fluorescent molecules important in biomedical analysis, including the fluorochromes Cascade Blue and Pacific Blue, the expressible fluorescent protein cyan fluorescent protein (CFP), and the fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazoline (ELF-97; for endogenous AP detection and cell surface labeling with AP-conjugated antibodies).

Methods: Comparisons were made between VLD excitation and a krypton-ion laser emitting at 407 nm (both at higher power levels and with the beam attenuated at levels approximating the VLD) on the same FACSVantage SE stream-in-air flow cytometer. We evaluated a Power Technology 408-nm VLD (30 mW) equipped with circularization optics (18 mW maximum output, set to 15 mW) and a Coherent I-302C krypton-ion laser emitting at power levels ranging from 15 to 75 mW.

Results: Cascade Blue, Pacific Blue, and CFP showed comparable signal-to-noise ratios and levels of sensitivity with VLD excitation versus the krypton-ion laser at high and VLD-matched power outputs. Multicolor fluorescent protein analysis with 488-nm excitation of green fluorescent protein and DsRed and VLD excitation of CFP was therefore feasible and was demonstrated. Similar levels of excitation efficiency between krypton-ion and VLD sources also were observed for ELF-97 detection.

Conclusions: These evaluations confirmed that VLDs may be cost- and maintenance-effective replacements for water-cooled gas lasers for applications requiring violet excitation in addition to DNA binding dyes.