🚀 go-pugleaf

    We've found that PE is an excellent acceptor for FITC energy transfer
    (and there are lot of commercially available monoclonals conjugated w/
    PE). But instead of measuring acceptor enhancement (since the PE is
    also excited by 488nm) we measure donor photobleaching.

    The method is described in:
    "Epitope mapping by photobleaching fluorescence resonance energy
    transfer measurements using a laser scanning microscope system"
    Biophys. J. 61:661-670 (92).

    Recent applications include:
    "The L-selectin (Leu8) molecule is associated with the TcR/CD3
    receptor; fluorescence energy transfer measurements on live cells"
    Immunol. Cell Biol. 72:319-324 (94). and,
    "Cross-linking of CD4 in a TCR/CD3-juxtaposed inhibitory state: a pFRET
    study" Biophys. J. 68:1170-1176 (95).

    These were done in a non-confocal mode (since we don't have that option
    on our Meridian ACAS 570 ... yet) and we actually expanded the beam to
    illuminate an entire lymphocyte and bleached at one position (since it
    is a stage scanning setup and the throughput on image photobleaching
    was too slow). If you have a beam scanning setup (video-rate would be
    good), you should be able to achieve the same effect by scanning an
    image of a single cell repeatedly over time.

    Hope this helps.

    Scott Pine
    FDA/Division of Research and Testing
    PINE@FDACD.CDER.FDA.GOV