Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis.
This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime.
Doornbos
R M
RM
Applied Optics Group, Faculty of Applied Physics, University of Twente, Enschede, The Netherlands.
De Grooth
B G
BG
Kraan
Y M
YM
Van Der Poel
C J
CJ
Greve
J
J
D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D007834:Lasers; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology; D010798:Phycocyanin; D011419:Propidium; D011806:Quinolinium Compounds; D013844:Thiazoles
D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D013844:Thiazoles; C002497:allophycocyanin; D010798:Phycocyanin; C075295:1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene)quinolinium; D011419:Propidium; D004247:DNA