Jim, All three at the same time will be a problem--especially if you use Cychrome--since there will be lots of fluorescent overlap between the FITC/PE pair and the PE/Cychrome pair. Best choice would be a combination of fluorochromes matched to specific excitation/emission filters in the filter cube, and (if you want to get pictures--highly recommended since memory can fade faster than FITC in the bright summer sun) a high resolution digital camera. That is FITC, PE, Texas Red would be good choices with the appropriate filters they give you single excitation/emission images--one for each fluorochrome--with no fluorescence overlap. That means, FITC would excite well with a 490 bandpass and emit around 520nm; PE would excite well around 515nm and emit around 575nm; and Texas Red (or the equivalent Alexa dye) would excite well around 568nm and emit well around 620nm. All should be doable with a Hg arc lamp, a Xe arc lamp, or even a W/halogen. So, you find a field of interest, compose the image, take three separate images (without moving slide), and do the various combinations of overlays in something like Photoshop. (N.B.: The absolute fluorescence emission--and their subsequent combined overlays--will have little meaning in terms of relative antigen content unless you use a proper calibration standard. And that's another topic altogether.) Dave ---------------- David M. Coder, Ph.D. email: dcoder1@qwest.net tel./messages: 206-499-3446 ----- Original Message ----- From: "jim phillips" jphillip@med.miami.edu To: cyto-inbox Sent: Friday, August 10, 2001 9:33 AM Subject: 3 color fluorescense on a Scope? > > Hello to everyone, > This is a little off the subject of flow... but I have an investigator > that want to do 3 color cell surface fluorescense on a microscope. The > question is .. what three fluorochomes can she use.. The microscope is > just a normal fluorescense scope. I know FITC and PE but is there a > third?? Can you use Cychrome??? She wants to view all three at the > same time.. any help would be appreciated.. Thanks... > Jim Phillips > University of Miami School of Medicine. > > ps.... I just finished my music CD .. entitled ... "222.... 2 old? 2 > fat? 2 ugly?" You can listen to it on mp3.. > > www.mp3.com/jim_phillips > >
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:28 EST