Martin, you were saying: > I wish to identify SP cells distinguished via their low fluorescence in the > blue (using a 450/20 BP filter) and the red (using a 675EFLP optical > filter) following excitation of Hoechst dye with a uv laser line. Currently > I'm using a Coherent Innova Enterprise with dual visible and uv lines, > however the uv power output (<60mwatts) is insufficient to optimally excite > the dye.... I don't think laser power is your problem. We have seen and sorted SP cells on a modified FACS II using 50mW UV from an I90. There was more than enough power; I believe we could have halved it. Our FACS II optics would be quite similar to your Vantage: Blue: filter 455DF20 + neutral density 0.5, PMT Hamamatsu R928, 640V, gain 1 Dichroic DM610SP Red: filter 660DF20, PMT Hamamatsu R1477, 640V, gain 8 1. LP red filter possibly better; we didn't have one available. 2. The neutral density in the blue and the gain 8 on the red are because we don't have independent Voltage controls and the blue signal is so bright. *However* we have also tried and *failed*, because of too little red signal, to see those cells on a MoFlo with the *same* optical filters and 50mW UV from an Enterprise 621 but with a less red-sensitive PMT (Cytomation's H957-06; a replacement H957-08 is in the pipeline). Maybe PMT sensitivity is your problem. Frank. Frank Battye \ / < The Cytometry Laboratory Dora Kaminaris \__/ <<<<< The Walter & Eliza Hall Institute Viki Lapatis ------!!<<<<<<<< Victoria 3050, AUSTRALIA Jennie Chan /!!\ <<<<< ph: 61_3_9345 2540 fax: 61_3_9347 0852 Catherine Tarlinton o !! \ < in:: "facs_copy@wehi.edu.au" Geza Paukovics / !! \
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