Analysis of violet-excited fluorochromes by flow cytometry using a violet laser diode.
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.
Telford
William G
WG
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. telfordw@mail.nih.gov
Hawley
Teresa S
TS
Hawley
Robert G
RG
D000469:Alkaline Phosphatase / Q000032:analysis / Q000528:radiation effects; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D005456:Fluorescent Dyes / Q000528:radiation effects; D049452:Green Fluorescent Proteins; D007726:Krypton; D007834:Lasers; D008164:Luminescent Proteins / Q000032:analysis / Q000528:radiation effects; D051379:Mice; D009942:Organometallic Compounds / Q000032:analysis / Q000528:radiation effects; D009943:Organophosphorus Compounds / Q000032:analysis / Q000528:radiation effects; D011799:Quinazolines / Q000032:analysis / Q000528:radiation effects; D052999:Quinazolinones
D015415:Biomarkers; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D011799:Quinazolines; D052999:Quinazolinones; C066850:Cascade Blue; D049452:Green Fluorescent Proteins; C079177:2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; D007726:Krypton; D000469:Alkaline Phosphatase