Hello Alex... I haven't done it myself, but I'm told that the 457 nm line generated by the Spectrum 70C might be a little marginal for good ECFP excitation on a stream-in-air sorter (Coherent spec's it at 30 mW). The labs I know of (including ours) that use the 457 nm line for CFP excitation generate it from more powerful argon-ion lasers, like the Coherent I-70C-2, -3, -4, or -5, or I-90 series. As you probably already know, CFP also excites well with violet lasers, either a krypton-ion source or a powerful (>20 mW) violet diode, if these options are available to you. At the emission end, both of your filters will work, but you may need some 488 nm laser blocking - 488 nm notch filters works well for this purpose. If ECFP has worked poorly using your Spectrum, adding the notch might help in spite of the low laser power. If you are doing CFP alone, you can also detect it through a GFP or FITC filter (510 - 530 nm) without the notch. Enjoy, Bill Telford NCI-NIH At 11:46 AM 3/15/2004 +0100, Alexander Schmitz wrote: >Dear Flowers, > >On a FACSVantage SE: >Is anybody using the 457 line of a Spectrum 70 laser as a secondary laser >to excite ECFP ? >The primary laser is 488nm. >What is the best filter ? 470/15 , 485/22 >Do I need other filters (eg. blocking filters ?) >Which beads can be used for the laser alignment ? >It would be great if you could share your experiences with me. >Many thanks >Alex > > >-- >______________________________________ >Dr. Alexander Schmitz >FACS Core Facility >Department for Molecular Biology (MBI) C.F. Moellers Allee 130/3 >DK 8000 Århus >Denmark >axs@mb.au.dk >Tel: +45 8942 2676 >Tel: +45 2338 2429 Mobil >Fax +45 8916 6500 >______________________________________ >Received on Tue Mar 16 14:38:00 2004
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