Fig. 6.
Cell imaging and single biomolecule tracking by complementation-induced intramolecular spFRET. (A) In vitro native gel shift assay of M3-A647 peptide binding to soluble GFP 1–10 (+) or in TGN buffer (−). The gel is sequentially imaged for M3-A647, GFP, and A647-GFP intramolecular FRET emission. (B) Live cell imaging of GFP 1–10-CD4 proteins complemented with fluorescent M3-A647 (Left) or nonfluorescent M3-biotin (Right) peptides and imaged by dual-color TIRF microscopy using only 488-nm excitation. Images are pixel-based maximum intensity projections of diffusing A647-GFP-CD4 proteins (ΣImax) for all frames of Movie S12. (Scale bar: 5 μm.) (C) GFP (green) and M3-A647 fluorescence time traces (red) along the diffusion path (Right) of individual A647-GFP-CD4 proteins showing intramolecular spFRET. Fluorescence background traces (gray) are taken in the immediate vicinity of the trajectories. The single-step photobleaching of GFP (Upper, green arrow) or M3-A647 (Lower, red arrow) induces an arrest of intramolecular spFRET (Movie S13). (D) Live cell TIRF imaging and tracking of individual A647-GFP-CD4 proteins by spFRET at high M3-A647 concentrations (0.7 μM) without washing. A cell is sequentially imaged using direct M3-A647 excitation at 638 nm (Left) and then, indirect spFRET excitation at 488-nm laser (Right). Individual A647-GFP-CD4 proteins diffusing in the plasma membrane can be tracked in the M3-A647 channel using indirect spFRET excitation (red trajectories and white squares) but are lost in the saturating surrounding fluorescent signal when directly excited at 638 nm (red squares). Three representative examples of A647-GFP-CD4 trajectories are presented (from white squares). (Scale bar: 10 μm.)