There are a number of differences between the two instruments, but the most significant difference is in the fluorescence collection optics. The sorter uses an objective lens to image the laser/stream intercept in air; the bench top analyzer has a similar objective lens but it is directly coupled with a gel to the cuvette to image the laser/stream intercept within. So, the analyzer has optics more like an oil immersion microscope objective and thus, captures more light the free air objective: More light means more sensitivity. There also some lesser differences in the filters (the total number and their transmission/reflection characteristics.) Remember that each filter is generally no better than 90% efficient (10% of the light is always lost), and the effect is multiplicative . That is, for three filters (i.e.,(.9*.9*.9)) leaves you with only 75% of the light collected by the objective lens. The FACScan has two filters in front of the FL3 detector; the Vantage may have more depending on your configuration. The most efficient filter for the fluorochrome may also help. (The 650LP in the FACScan may let more Cy5 emission through the 675BP20). Dave =================== David M. Coder, Ph.D. Consultant in Cytometry Seattle, Washington tel./message: 206-499-3446 email: d_coder@msn.com -----Original Message----- From: Eckstein, Volker [mailto:Volker.Eckstein@med.uni-heidelberg.de] Sent: Monday, November 18, 2002 8:43 AM To: cyto-inbox Subject: Fluorescence sensitivity FACScan vs. FACS Vantage SE Hi collegues, is there anyone who can tell me something about the differences of fluorescence sensitivity on the FACScan and the FACS Vantage SE? I had a badly labelled sample of white blood cells labelled with CD4-CyChrome (PECy5). On the Vantage equipped with a 100um nozzle it was impossible to detect any fluorescence on FL3-channel with a 675/20 BP-filter at 100mW argon laser light. After relabelling a small aliquot of the cells with the same antibody (10ul antibody on 4x10exp6 cells) I could detect a dim fluorescence. Not even a log between the negatives and the positives but good enough for a calculation. I have used antibodies with CyChrome several times before with other cells and never have seen such a bad fluorescence. All the optics were well aligned. But as I said I think the amount of antibody (100ul antibody solution in a 5ml volume of PBS with 2%FCS for 2x10exp8 white blood cells) is not sufficient for proper sorting. Interestingly the CD4-CyChrome bad labelled cells could easily be seen on the Fl3-channel on our old old FACScan. Even with this little amount of antibody there was a clear and beautiful difference between CD4pos. and neg. cells. The relabelled aliquot of cells didn't give any improved signal on the FACScan. Any suggestions? Thanks in advance Best regards Volker Volker Eckstein PhD Dept. of Internal Medicine V Medical school of the university University of Heidelberg GERMANY volker_eckstein@med.uni-heidelberg.de
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:30 EST