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

Authors


  • This article is a US Government work and, as such, is in the public domain in the United States of America.

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. Cytometry Part A 54A:48–55, 2003. Published 2003 Wiley-Liss, Inc.

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