Figure 3. Raw image data of single fluorescently labeled Tom40 molecules.

(A, E) Differential interference contrast (DIC) image of an isolated S. cerevisae mitochondria immobilized on a poly-L-lysine coated coverslip. (B, F) Auto-fluorescence image acquired during the photoconversion pulse. (C, G) Overlay of DIC and autofluorescence images. (D) 100 consecutive frames acquired during Tom40 tracking are displayed in a single frame, using maximum projection. Displayed in color are the resulting Tom40 trajectories. The right panel depicts the trajectories (in color) in a zoomed-in 5×5 pixel area (500 nm scale bar). (H) Maximum projection of seven consecutive frames acquired during tracking of an individual Tom40 molecule. Displayed in color is the resulting Tom40 trajectory. The right panel displays a zoomed-in region of the resulting trajectory (3×3 pixel area, 250 nm scale bar). (I–Q) Tracking of an individual Tom40 molecule in the mitochondria depicted in (E–H). Fluorescence images showing single-step photoconversion (I–J), diffusion (J–P) and single-step photobleaching (P–Q) of a single Dendra2-labeled Tom40 molecule. All images are taken with a frame time of 5 ms and an exposure time of 5 ms, and correspond to time 0 to 40 ms, as indicated on the image. The scale bar represents 1 μm. The intensity scalebar for all 16-bit fluorescence images is depicted in the lower right corner.