The Vantage is configured with a Coherent INNOVA Enterprise Model 621 argon ion laser emitting simultaneously at 488 nm (150 mW maximum output) and 351 nm (50 mW maximum output) and a SpectraPhysics Model 127 Stabilite helium neon laser emitting at 632 nm (25 mW maximum output). A Coherent INNOVA 90 krypton ion laser can also be configured into the optical path in place of the helium neon laser. The argon 488 nm beam intercepts the stream at the primary position, and the 351 nm UV and 632 nm helium neon beams intercept at the secondary position. Three PMTs are configured to analyze signals from the primary laser and two from the secondary. Signals from both beams are split using a 610 short pass dichroic for the 488 nm-originating signal (passing signals to PMT1 and PMT2, and reflecting signal right to PMT3) and a mirror for the UV/HeNe-originating signals (reflecting signal left to PMT4 and PMT5). PMT1 and PMT2 are usually used to analyze FITC (yellow-green) and PE (orange) respectively, their signals separated by a 560 short pass dichroic. PMT3 is usually used to analyze PE-Cy5 conjugates or PerCP (red); this signal is reflected to the right using the 610 short pass dichroic. PMT4 and PMT5 are usually used to analyze APC (red) and Hoechst 33258 or AMCA (blue) respectively; their signals are separated using a 640 long pass dichroic. FITC, PE, PerCP, APC and Hoechst 33258/AMCA signals are ultimately passed through 535/30, 575/24, 675/20, 660/20 and 424/44 narrow bandpass filters, respectively, before reaching the PMTs. All PMT configurations can be modified, within the restrictions of the primary 488 nm and secondary 351/632 nm signal paths. For example, PMT4 and PMT5 can be configured with a 530 long pass dichroic and 405/20 and 485/22 narrow bandpass filters to detect the fluorescent calcium indicator indo-1. PMT3 can also be fitted with a 610/20 narrow bandpass filter to detect PE-Texas Red conjugates such as RED613.

To see a 5-color experiment with the Vantage, click here.

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