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. 2012 Mar 1;23(5):918–929. doi: 10.1091/mbc.E11-07-0622

FIGURE 1:

FIGURE 1:

Single-molecule detection of newly synthesized Venus-ARC protein molecules in injected cells. (A) Experimental procedure. Fluorescently labeled RNA molecules (green/red squiggles) encoding Venus-ARC or Venus-FMRP were microinjected into the perikaryon of cultured hippocampal neurons. Microinjected RNA molecules assemble into RNA granules (light red circles) that are transported along dendrites to dendritic spines, where translation occurs. Newly synthesized Venus fusion protein molecules undergo fluorophore maturation and are detected by single-molecule imaging. (B) Time-lapse images (100 frames, 200 ms/frame, total time 20 s) of a single region of interest (ROI; 2.5 × 2.5 μm) within a dendrite. Four temporal trajectories corresponding to four separate translational events within the ROI are detected in the sequence shown. The initial frame of each trajectory is outlined in green. (C) Normalized fluorescence intensity trajectories for molecules corresponding to trajectories 1 and 4 in A illustrating single-step appearance, photobleaching, and blinking, consistent with single-molecule behavior. (D) Histogram of photobleaching coefficients (PBs) for single-molecule trajectories. (E) Distribution of photobleaching times for fluorescent molecules. The solid curve represents the fit to a single-exponential decay.