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Figure 2


Fig. 2. Single-molecule imaging of Crac-GFP in living D. discoideum cells. (A) Configuration of a total internal reflection fluorescence microscope (TIR-FM) for single-molecule imaging attached to an inverted microscope (IX-70, Olympus Inc, Japan). (B) Schematic illustration of single-molecule imaging of Crac-GFP bound to the basal membranes of a D. discoideum cell that is illuminated by evanescent fields generated by TIR of excitation light on the surface of a coverslip. (C) Typical images of Crac-GFP visualized by TIR-FM (left panel). Fluorescent spots were hardly detected in a cell expressed GFP alone (right panel). (D) Histogram of fluorescence intensities of the spots of Crac-GFP observed in living cells (dark gray bars) and on the surface of coverslips (light gray bars). Each distribution was fitted to an Gaussian function. (E) An example of a single-step photobleaching of Crac-GFP on coverslips. (F) An intensity profile of a fluorescent spot representing a single Crac-GFP molecule attached to a coverslip. (G) An example of a single-step photobleaching of Crac-GFP in fixed cells. (H) An intensity profile of a fluorescent spot representing a single Crac-GFP molecule in a fixed cell. The background fluorescence was heterogeneous when compared to (F), which came from unbound Crac-GFP molecules present in the cytosol.





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