Solid state 488 nm excitation on the LSR II.
The LSR II uses a Coherent Sapphire 20 mW solid state 488 nm laser, replacing the air-cooled argon-ion gas lasers found on the original LSR and earlier flow cytometers. These lasers have minimal cooling requirements and a reputed to be highly stable; plasma tube glow and noise is also eliminated.
(Below). The Coherent Sapphire 25 mW solid state 488 nm laser. The laser is the silver unit in the bottom half of the photo; the black object on top of it is a (largely unnecessary) heat sink. The beam shaping optics (required to match the beam diameter of the violet diode laser) are aiming to the right.
Single laser four-color analysis on the LSR II. The 488 nm laser "octagon" is currently equiped with five PMTs, four for fluorochrome detection and one for side scatter mesasurement. The unit can accomodate up to seven PMTs, six for fluorochrome analysis. The octagon uses a series of long pass dichroics to transmit and reflect incoming signals, "capturing" the longest fluorochrome signals (such as PE-Cy7) in the proximal PMTs and the shortest wavelength signals (such as FITC and side scatter) in the distal PMTs. The default configuration is set up for simultaneous FITC, PE, PerCP-Cy5.5 and PE-Cy7 analysis; the more common fluorochrome PE-Cy5 can also be substituted for PerCP-Cy5.5 with this configuration.
(Below). "Octagon" configuration for the primary 488 nm detectors. PMTs are contained in the vertical cylinders; filters and dichroics are visible in slots positioned around the center of the unit. The transmitting fiber optic enters the unit from the left.
(Below). The current default configuration for the primary PMT "octagon". This configuration allows four-color analysis of FITC, PE, PerCP-Cy5.5 and PE-Cy7 off the primary 488 nm solid laser. The two "distal" detector slots in the signal path have no PMTs or dichroics.
(Below). Four-color analysis of FITC, PE, PerC-Cy5.5 and PE-Cy7 using the default "octagon" configuration. EL4 cells were labeled with biotin-conjugated CD44 followed by FITC, PE or PerCP-Cy5.5 and PE-Cy7-conjugated streptavidin, and a "cocktail" of unlabeled / FITC / PE / PerCP-Cy5.5 / PE-Cy7 labeled cells was analyzed using the above default configuration.
(Below). Four-color analysis of FITC, PE, PE-Cy5 and PE-Cy7 using the default "octagon" configuration. EL4 cells were labeled with biotin-conjugated CD44 followed by FITC, PE or PE-Cy5 and PE-Cy7-conjugated streptavidin, and a "cocktail" of unlabeled / FITC / PE / PerCP-Cy5.5 / PE-Cy7 labeled cells was analyzed using the above default configuration.
The octagon can be modified for better analysis of PE-Cy5 by inserting a 670/14 nm bandpass filter in front of the PE-Cy5 detector (in place of the 695/40 filter used for PerCP-Cy5.5) and replacing the corresponding 680 LP dichroic with a 635 LP (custom fabricated by Omega Optical).
(Below). The modified configuration for the primary PMT "octagon". The PerCP-Cy5.5 595/40 nm filter has been replaced with a 670/14 nm PE-Cy5 filter, and the corresponding dichroic has been replaced with a 635 LP. This configuration allows better four-color analysis of FITC, PE, PE-Cy5 and PE-Cy7 off the primary 488 nm solid laser.
(Below). Four-color analysis of FITC, PE, PE-Cy5 and PE-Cy7 using the modified "octagon" configuration. EL4 cells were labeled with biotin-conjugated CD44 followed by FITC, PE or PE-Cy5 and PE-Cy7-conjugated streptavidin, and a "cocktail" of unlabeled / FITC / PE / PerCP-Cy5.5 / PE-Cy7 labeled cells was analyzed using the above modified configuration.
DNA content analysis on the LSR II. Measurement of cellular DNA content and cell cycle is traditionally carried out with the DNA binding dye propidium iodide (PI) on flow cytometers equipped with quartz flow cells. The good light collection efficiency and low numerical aperture of the LSR II collection optics suggests that it might be well-suited for DNA content analysis using PI. The LSR II was therefore tested for DNA content analysis using chicken RBCs, trout RBCs and calf thymus nuclei standards, as well as EL4 cells labeled with PI. The LSR II gave some improvement in DNA content peak C.V.s compared to the BD FACSCalibur, an indicator of cell cycle resolution.
(Below). DNA content analysis with propidium iodide on the LSR II. Chicken red blood cells (CRBCs), trout red blood cells (TRBCs) and calf thymus nuclei (CTNs) were labeled with PI at 50 ug/ml with simultaneous RNase treatmentand analyzed on the LSR II with the 488 nm laser and through the PE detector (left column). Cells were simultaneously analyzed on the BD FACSCalibur with 488 nm excitation and signal collection through the PE channel (right column). Mean fluorescent intensities and C.V.s for the singlet peaks are shown.
(Below). DNA content analysis with propidium iodide on the LSR II. EL4 cell nuclei were prepared with a solution of NP-40 in PBS and labeled with PI at 50 ug/ml with simultaneous RNase treatment, followed by analysis on the LSR II with the 488 nm laser and through the PE detector (left histogram). Cells were simultaneously analyzed on the BD FACSCalibur with 488 nm excitation and signal collection through the PE channel (right histogram). Mean fluorescent intensities and C.V.s for the singlet peaks are shown.
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