Extended Data Fig. 5: FRET assay using confocal microscopy.
From: Multimodal probing of T-cell recognition with hexapod heterostructures
a, Photobleaching of CD19-conjugated AF555-hexapod substantially increases the fluorescence of CAR conjugated GFP. Shown from left to right are excited donor (GFP-anti-CD19 CAR) images before bleaching, excited acceptor (AF555-CD19-hexapod) images before bleaching, excited donor images after bleaching, excited acceptor images after bleaching, calculated FRET efficiency images (pseudo-color, where a yellow color spectrum represents strong to weak FRET efficiency), and differential interference contrast (DIC) images. b, Substituting CD19 with anti-CD45, an antibody that links the hexapod to CAR-T cell by binding with CD45 on the cell surface (instead of CD19 CAR binding activity), significantly decreased the FRET efficiency. The experiments were conducted in the same manner as in a, except AF555-CD19-hexapod was replaced with AF555-anti-CD45-hexapod. Scale bar, 5 μm. The experiment was repeated independently for five times with similar results.
