From: Dave Coder (dave@nucleus.immunol.washington.edu)
Date: Fri Jul 09 1993 - 12:30:17 EST
Polarization on the FACStarPlus Fluorescence polarization on the FACStarPlus is fairly easy to setup and measure. There are two issues involved: 1. optics (getting, installing, and aligning) 2. data analysis: computing polarization from detector pulse height measurements. Optics: We've done fluorescence polarization using 488nm excitation. Optics include a 520LP filter (to remove 488nm side scatter), a 50/50 beam splitter (placed in the dichroic filter position between detectors FL1 and FL2) to separate vertical and parallel components, and polarizers in front of FL2 (vertically-oriented with respect to the plane of the laser) and FL1 (horizontally oriented). The optical system was balanced using a half wave retarder in a rotation mount to adjust the vertical plane of the laser to 45 degrees, and adjusting PMT high voltages such that the response from both detectors was equal. (The half wave plate was mounted on an aluminum plate fixed to one of the laser cover mounting positions in front of the 2W argon laser. Yes, the laser safety covers are removed. The cover mount on the cast optical bench is non-precision (that is, it is not parallel to the plane of the bench) so shims were used to get the half wave plate vertical plane perpendicular.) Optics were purchased from Newport Optical (Fountain Valley, California) except for the 50/50 beam splitter mounted in an aluminum ring (special order; the beam splitter is very thin and fragile, you want it mounted in a ring) from Omega Optical (Brattleboro, Vermont). The beam splitter in its mounting ring was mounted in a custom-machined filter holder (working drawings available as a device-independent PostScript file by anonymous ftp at flowcyt.cyto.purdue.edu; see following article for instructions on downloading.) Computation of Polarization Fluorescence polarization is the difference of the parallel and perpendicular components divided by the sum of the two. Computing this value for each cell needs to be done using the parallel and perpendicular list mode values since there are no analog processing electronics on the FACStarPlus. There are several ways to compute the polarization values from list mode data. We accomplished this using a simple package in Mathematica which computes the polarization for each value (multiplied by a suitable constant for proper scaling), and writes a new list mode file with the additional derived parameter. A brief summary was presented in a poster at the last ISAC meeting. Dave Coder Dept. of Immunology Univ. of Washington Internet: dcoder@u.washington.edu fax: 206-543-3480 tel: 206-685-3014
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