Frequency-tripled Nd:YVO₄ UV laser on the BD LSR II.

Lightwave Electronics, in collaboration with BD Biosciences, recently developed a frequency-tripled Nd:YVO₄ solid state ultraviolet laser for the BD LSR II.  Although technically a pulsed laser source, this laser has a sufficiently short pulse interval to operate essentially as a quasi-CW laser, allowing its use on a flow cytometer.  Unlike most commercially developed Nd:YVO₄ UV sources, this laser is of relatively low power (~20 mW), applicable for use on a cuvette-equipped flow cytometer.  As a UV source, the Nd:YVO₄ gave excellent results using our microsphere sensitivity and resolution standards, slightly better than that seen with our NUVLD source.  It gave Hoechst 33342 side population comparable to the NUVLD source, and good indo-1 performance (an application not possible with the NUVLD source).

(Below).  Lightwave Electronics Nd:YVO₄ UV laser, temporary installation.  The mode-locked UV laser was mounted temporarily on the BD LSR II prior to permanent installation, allowing laser alignment with two steering UV-compatible steering mirrors (detail in the lower photo).   In this install, the laser was aligned to the third PMT trigon (normally used with the violet laser diode).

(Below).  Details on the steering mirrors.

(Below).  Analysis of microsphere sensitivity/resolution standards with the UV laser.  Left histograms, Polyscience 2 micron yellow-green microspheres were analyzed on the BD LSR II with either a NUVLD (374 nm, 8 mW) or the Nd:YVO₄ (355 nm, 22 mW).  At the same PMT gain settings and filter configuration, the fluorescence signal was roughly equivalent for both laser sources.   Middle histograms, Molecular Probes InSpeck Blue microspheres were analyzed using either the NUVLD or the Nd-YAG.  Both lasers gave excellent resolution of all seven differentially labeled bead populations.  Right histograms, When the dimmest (0.3% and unlabeled  InSpeck beads were analyzed with both lasers with linear scaling, the Nd:YVO₄ laser gave slightly better resolution based on the peak C.V. of the dimmest bead population.

(Below).  Analysis of microsphere sensitivity/resolution standards with the Nd:YVO₄ laser.  Left histograms, Spherotech Rainbow 8-population microspheres were analyzed on the BD LSR II with either a NUVLD (374 nm, 8 mW) or the Nd:YVO₄ (355 nm, 22 mW) through a 450/50 nm filter.  All eight populations were resolvable with both lasers, although the Nd:YVO₄ gave slightly better resolution of the separation between the two dimmest populations (right histograms).

(Below).  Hoechst 33342 side population analysis with the Nd:YVO₄ laser.  Mouse bone marrow was labeled with Hoechst 33342 at 5 ug/ml for 90 minutes and analyzed on the BD LSR II using the Nd:YVO₄ laser.  The Hoechst side population was easily resolvable with this laser source, comparable to the NUVLD.

(Below).  Indo-1 analysis on the BD LSR II with the Nd:YVO₄ laser.   Mouse EL4 cells were loaded with the ratiometric calcium indicator indo-1 AM at 5 ug/ml for 45 minutes and analyzed on the BD LSR II with either a NUVLD (374 nm, 8 mW) or the Nd:YVO₄ (355 nm, 22 mW), using 405/20 and 530/30 nm filters and a 450 LP splitter.   Ionomycin at 2 uM was used to stimulate calcium uptake at 1 minute following initiation of event collection.   The NUVLD, due to its longer wavelength, as not applicable for indo-1 analysis; however, the Nd:YVO₄ gave good results

(Below).  Lightwave Electronics Nd:YVO₄ UV laser, final installation.  The BD install of the Nd:YVO₄ is shown below.  Since there is minimal beam alignment possible with the factory install, the flow cell mest be aligned to the UV beam, and all other lasers subsequently aligned to the flow cell.

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