The absorption of the Alexa Fluor 350 conjugate is reasonably well excited by the UV argon-ion laser at 351-363 nm so that is not the problem, nor is photobleaching. http://iw.probes.com/servlets/spectra?fileid=11045ph8 However, this points out a general problem of trying to correlate fluorescence microscopy and flow cytometry that may be the problem here too. In microscopy one can sometimes visualize signals only because the fluorescence is concentrated in relatively small regions of the cell. In flow cytometry the fluorescence measured is from the entire cell. For a weak signal -- such as UV-excited reactive dyes -- the autofluorescence of the cell, particularly in the UV, can be greater than the specific signal, particularly in the UV. The autofluorescence in microscopy may be dispersed thinly, or almost invisible to the eye (or film) but the volume of the cell may make the TOTAL autofluorescence higher than the specific signal. I suspect that that is what is happening here if you can see it in imaging but not flow. We have been working on amplifying the signal in flow cytometry using tyramide signal amplification (TSA) to increase the specific signal well beyond the autofluorescence, particularly for detecting low-abundance receptors. Although we have not yet done this in flow using UV-excited fluorophores such as the Alexa Fluor 350 dye, it has worked very well with longer-wavelength dyes where the specific signal was not detected over the autofluorescence, as in Panel B on the EGF receptor in 3T3 cells in thsi link. http://www.probes.com/handbook/figures/1487.html We have prepared TSA kits containing Alexa Fluor 350 tyramide that may amplify the UV signal well above autofluorescence. http://www.probes.com/handbook/sections/0603.html We also had a poster at the recent Cell Biology meeting doing this for estrogen receptors in flow. Those data are not yet in linkable form. "Braun, Ruedi" wrote: > Hi all, > I have a question concerning the use of Alexa 350, bound to a monoclonal Ab. > On a flourescence microscope, we could detect stained cells. However, on the > flow cytometer (MoFlo with a Coherent I90C UV Laser), I could not detect a > signal. Molecular Probes suggested, that the energy (100mW) of the Laser > would bleach the flourochrome and therefore no signal is detected. Is this > possible? Or is the Argon Laser bleaching the compound? > The system has two lasers, 488 nm Argon Laser at 100mW on position 1 and the > UV Laser on position 3 (refering to the pinholes). The Filters for the > detection of Alexa 350 was 450/65 of a dichroic 510LP mirror. > Thanks for any hints. > Merry Christmas and Happy New Year! > Ruedi > > Dr. Ruedi K Braun > Ottawa Health Research Institute > 501 Smyth Rd > Ottawa, ON K1H 8L6 > Canada > > phone: (613) 737-8899 ext 73916 > e-mail: rbraun@ohri.ca
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:44 EST