Near-ultraviolet laser diodes for brilliant ultraviolet fluorophore excitation

Abstract

Although multiple lasers are now standard equipment on most modern flow cytometers, ultraviolet (UV) lasers (325-365 nm) remain an uncommon excitation source for cytometry. Nd:YVO4 frequency-tripled diode pumped solid-state lasers emitting at 355 nm are now the primary means of providing UV excitation on multilaser flow cytometers. Although a number of UV excited fluorochromes are available for flow cytometry, the cost of solid-state UV lasers remains prohibitively high, limiting their use to all but the most sophisticated multilaser instruments. The recent introduction of the brilliant ultraviolet (BUV) series of fluorochromes for cell surface marker detection and their importance in increasing the number of simultaneous parameters for high-dimensional analysis has increased the urgency of including UV sources in cytometer designs; however, these lasers remain expensive. Near-UV laser diodes (NUVLDs), a direct diode laser source emitting in the 370-380 nm range, have been previously validated for flow cytometric analysis of most UV-excited probes, including quantum nanocrystals, the Hoechst dyes, and 4',6-diamidino-2-phenylindole. However, they remain a little-used laser source for cytometry, despite their significantly lower cost. In this study, the ability of NUVLDs to excite the BUV dyes was assessed, along with their compatibility with simultaneous brilliant violet (BV) labeling. A NUVLD emitting at 375 nm was found to excite most of the available BUV dyes at least as well as a UV 355 nm source. This slightly longer wavelength did produce some unwanted excitation of BV dyes, but at sufficiently low levels to require minimal additional compensation. NUVLDs are compact, relatively inexpensive lasers that have higher power levels than the newest generation of small 355 nm lasers. They can, therefore, make a useful, cost-effective substitute for traditional UV lasers in multicolor analysis involving the BUV and BV dyes.

Keywords: NUVLD; brilliant ultraviolet dye; flow cytometer; near-ultraviolet laser diode; ultraviolet laser.

Figure 1.

Optical configuration for BD Biosciences LSR II used in this study. Two PMT trigon clusters were aligned with either the UV 355 nm or NUVLD 375 nm laser, and a violet laser diode emitting at 405 nm. Filters and dichroics are shown and were configured as described in the Materials and Methods section.

Figure 2.

Comparison of BUV395, BUV496, and BUV737 excitations using labeled mouse splenocytes and either the UV 355 nm laser (top three rows) or NUVLD 375 nm laser (bottom row). Gray traces depict unlabeled cells, and black traces depict labeled cells. Filters are as indicated in Figure 1 and the Materials and Methods section. The staining index for each positive peak is indicated in each histogram. Staining indices for BUV 395 anti-mouse CD4 and BUV496 anti-mouse CD45R are for the positive peak only, and BUV737 anti-mouse CD44 is for the entire population.

Figure 3.

(a) Optical configuration for determining incidental excitation of BV421 and BV510 by UV laser source. (b) Excitation of BV421 and BV510 labeled VersaChrome compensation microspheres by the UV 355 nm laser (top dotplot), NUVLD 375 nm (middle dotplot), or violet laser diode 405 nm (bottom dotplot). Staining indices for each axis are shown to the left of each dotplot.

Figure 4.

Five-color labeling of mouse splenocytes using directly conjugated antibodies with BUV395, BUV496, BUV737, BV421, and BV510. (a) Analysis with UV 355 nm laser and a 390/18 nm filter for BUV395. (b) Analysis with UV 355 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. (c) Analysis with NUVLD 375 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. Spillover matrices for each analysis are shown below the dotplots.

Figure 4.

Five-color labeling of mouse splenocytes using directly conjugated antibodies with BUV395, BUV496, BUV737, BV421, and BV510. (a) Analysis with UV 355 nm laser and a 390/18 nm filter for BUV395. (b) Analysis with UV 355 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. (c) Analysis with NUVLD 375 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. Spillover matrices for each analysis are shown below the dotplots.

Figure 4.

Five-color labeling of mouse splenocytes using directly conjugated antibodies with BUV395, BUV496, BUV737, BV421, and BV510. (a) Analysis with UV 355 nm laser and a 390/18 nm filter for BUV395. (b) Analysis with UV 355 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. (c) Analysis with NUVLD 375 nm laser and a 405/30 nm filter with 405 nm notch filter for BUV395. Spillover matrices for each analysis are shown below the dotplots.

Figure 5.

(a) Optical configuration for LSR II for simultaneous analysis of BUV737, BUV805, BV711, and BV785. (b) Comparison of BUV737 and BUV805 excitation using labeled mouse splenocytes and either the UV 355 nm laser (top row) or NUVLD 375 nm laser (bottom row) using the above filters. Left column BUV737 anti-CD4; middle and right columns, BUV anti-mouse CD3 and CD20 labeling. Gray traces depict unlabeled cells and black traces depict labeled cells. The staining index for each positive peak is indicated in each histogram. Staining indices for each labeling are shown. (c) Four-color labeling of human PBMCs using directly conjugated antibodies with BUV737, BUV805, BV711, and BV785 using UV 355 nm laser (top dotplots) or NUVLD 375 nm laser (bottom dotplots).

Figure 5.

(a) Optical configuration for LSR II for simultaneous analysis of BUV737, BUV805, BV711, and BV785. (b) Comparison of BUV737 and BUV805 excitation using labeled mouse splenocytes and either the UV 355 nm laser (top row) or NUVLD 375 nm laser (bottom row) using the above filters. Left column BUV737 anti-CD4; middle and right columns, BUV anti-mouse CD3 and CD20 labeling. Gray traces depict unlabeled cells and black traces depict labeled cells. The staining index for each positive peak is indicated in each histogram. Staining indices for each labeling are shown. (c) Four-color labeling of human PBMCs using directly conjugated antibodies with BUV737, BUV805, BV711, and BV785 using UV 355 nm laser (top dotplots) or NUVLD 375 nm laser (bottom dotplots).