Violet laser diodes as light sources for cytometry.
BACKGROUND
Violet laser diodes have recently become commercially available. These devices emit 5-25 mW in the range of 395-415 nm, and are available in systems that incorporate the diodes with collimating optics and regulated power supplies in housing incorporating thermoelectric coolers, which are necessary to maintain stable output. Such systems now cost several thousand dollars, but are expected to drop substantially in price. Materials and Methods A 4-mW, 397-nm violet diode system was used in a laboratory-built flow cytometer to excite fluorescence of DAPI and Hoechst dyes in permeabilized and intact cells. Forward and orthogonal light scattering were also measured.


RESULTS
DNA content histograms with good precision (G(0)/G(1) coefficient of variation 1.7%) were obtained with DAPI staining; precision was lower using Hoechst 33342. Hoechst 34580, with an excitation maximum nearer 400 nm, yielded the highest fluorescence intensity, but appeared to decompose after a short time in solution. Scatter signals exhibited relatively broad distributions.


CONCLUSIONS
Violet laser diodes are relatively inexpensive, compact, efficient, and quiet light sources for DNA fluorescence measurement using DAPI and Hoechst dyes; they can also excite several other fluorescent probes.
Shapiro
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Howard M. Shapiro, M.D., P.C., West Newton Massachusetts 02465-2513, USA. hms@shapirolab.com
Perlmutter
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D002467:Cell Nucleus; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D007834:Lasers; D008027:Light; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D004247:DNA