Near UV laser diodes (NUVLDs) on the BD LSR II.
Near-UV GaN diode lasers (NUVLDs) are recent developments in diode laser technology. These lasers emit in the 370 to 380 nm range, approaching true ultraviolet emission. Like more widely available blue and violet diodes, they are small, air-cooled and are becoming commercially available in increasing power levels. These lasers have the potential to provide a source of near-ultraviolet excitation in a package far smaller and lower in maintenance than other UV lasers, such gas sources and triple-pumped Nd-YAG lasers. While violet diodes (395 to 410 nm) can substitute for certain UV applications (such as DAPI cell cycle), laser sources closer to true UV are likely to provide improved cell cycle analysis resolution. In addition, Hoechst 33342 side population (SP) analysis is poorly resolved by violet excitation; near-UV sources may provide better SP resolution, closer to that obtained with traditional UV-emitting gas lasers. NUVLDs are also likely to make good excitation sources for UV-excited immunophenotyping fluorochromes like Alexa Fluor 350 and Marina Blue, as well as recently developed Quantum Dots. To test the utility of NUVLDs for flow cytometry, several NUVLDs were mounted on our BD LSR II and evaluated for their ability to excite a variety of UV fluorochromes.
Incorporation of NUVLDs onto the BD LSR II. Four NUVLDs were mounted on the LSR II: a Power Technology 372 nm emitting at 1.5 mW, a Power Technology 370 nm emitting at 8 mW, a Coherent Radius 372 nm emitting at 8 mW, and a Point Source Ltd. 374 nm emitting at 10 mW. These lasers were mounted in the front-most laser position on our LSR II (in the UV position) and their beams aligned to the violet laser pinhole (the second signal path) on the LSR II. Power levels were confirmed with a Melles Griot broadband power meter. Laser and mounts are shown below. Lasers were aligned using Polyscience 2 micron yellow-green microspheres.
(Below). Power Technology 370 nm 8 mW laser.
(Below). Coherent 372 nm 8 mW laser.
(Below). Point Source Ltd. 374 nm 10 mW laser. Shown and used here without a fiber optic.
Alignment and sensitivity assessment. Laser alignment and resulting signal sensitivity was measured using Molecular Probes InSpeck Blue microspheres. For comparison purposes, the same microspheres were analyzed on a FACSVantage DiVa equipped with a krypton-ion laser emitting in the multiline UV mode at 100 mW. As shown below, the NUVLD emitting at 1.5 mW gave reduced sensitivity for the dimmest bead populations compared to the gas laser; however, the more powerful versions (8-10 mW) gave sensitivity levels comparable to or better than the gas source, easly distinguishing the dimmest bead population from the unlabeleds. As prevously observed with violet diodes, the combination of low-power diode source and a cuvette-confined sample stream gave results comparable to a gas laser emitting at a much higher power level on a stream-in-air instrument. All NUVLD 8-10 mW sources performed comparably.
(Below). Sensitivity assessment of NUVLD sources on the BD LSR II. Molecular Probes InSpeck Blue microspheres were analyzed on the BD LSR II equipped with the indicated laser source.
DAPI DNA content analysis. The ability of the NUVLDs to carry out DAPI DNA cell cycle was then assessed. Chicken red blood cell (CRBC), trout red blood cell (TRBC) and calf thymus nuclei (CTN) DNA standards labeled with DAPI were analyzed, as well as EL4 mouse thymoma cell nuclei. The 1.5 mW source gave acceptable DNA resolution, comparable to that obtainable with propidium iodide; however, the 8-10 mW versions were a significant improvement, giving peak C.V.s well below 2.0 for all DNA standards tested. Resolution was better than that previously observed for a more powerful violet diode source on the same instrument (see data here).
(Below). DNA content analysis using NUVLDs on the BD LSR II. Top histogram, CRBCs analyzed with the NUVLD 1.5 mW. Bottom four histograms, CRBCS, TRBCs, CTNs and EL4 mouse thymoma nuclei analyzed with an NUVLD 8 mW. Peak C.V.s are indicated by each peak.
Immunophenotyping fluorochromes. The UV-excited immunophenotyping fluorochromes Alexa Fluor 350 and Marina Blue (both structurally based on the coumarin probe 7-aminomethylcoumarin or AMCA) are useful if somewhat dim probes for multicolor analysis. The fluorogenic alkaline phosphatase substrate ELF-97 is a much brighter UV-excited probe that can be used in same cases as an immunophenotyping label. NUVLDs in the 8-10 mW range gave sensitivity levels for these probes comparable to the gas laser on the FACSVantage DiVa.
(Below). Immunophenotyping probe excitation with NUVLDs. EL4 cells were labeled with either biotin-conjugated anti-CD44 or CD90 followed by streptavidin-conjugated Alexa Fluor 350 or Marina Blue, or streptavidin-conjugated alakline phosphatase followed by the ELF-97 substrate. Cells were then analyzed on either the LSR IIwithone of the NUVLD sources (top row), or the FACSVantage DiVa with the krypton-ion MLUV laser (bottom row).
Quantum dots. Quantum dots, encapsulated complexes of inorganic salts, are proving to be very useful fluorescent probes for a variety of applications. Since these probes are particularly well-excited by shorter wavelength lasers, we tested them with the NUVLD sources. In contrast to the low molecular weight probes shown above, sensitivity for Qdot fluorescence was better on the LSR II with all the 8-10 mW NUVLD sources, compared to the FACSVantage with the gas laser.
References
Telford, W.G. Small lasers in flow cytometry (invited book
chapter). In Flow Cytometry, 2nd
Edition, Humana Press, London, UK.
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