Fig. 1: Development of single-molecule microscopy in live hair cells.

a Our workflow for single-molecule microscopy. b Labeling density optimization using vestibular hair cells expressing HaloTag-actin. At 0.3 nM JFX554-ligand, the entire cells are labeled, with dense signal at stereocilia tips (arrows) and in the cuticular plate. Fluorescent puncta appear in the cell body at 0.1 nM (arrowheads) and in stereocilia around 0.01 nM (open arrowheads). Maximum projections of volume scans. Exposure, 100 ms per plane at 0.2 kW/cm². Bars, 5 µm. c Classification of fluorescent puncta using the Gaussian Mixture model⁶⁰. The sum intensity is calculated by integrating the pixel values after background subtraction. Among the three populations (Pop1, Pop2 and Pop3), the peak intensity of Pop2 (985) is approximately twice that of Pop1 (408), indicating that Pop1 and Pop2 are emitted from one and two fluorophores, respectively. A total of 76 puncta were analyzed from six cells (including b, 0.01 nM), using average projections of 12 planes per volume scan. d Comparison between the average line intensity profile of fluorescent puncta (orange solid line) and the theoretical point spread function (PSF) of the objective lens calculated using the Born & Wolf 3D Optical Model^117,118 (black dashed line). The similarity between these two intensity curves suggests that these puncta are emitted from a point source. Fluorescence intensity is an average of 10 puncta in b (0.01 nM, Pop1 only). SD, orange dotted lines. e, f Representative kymograms of non-fused HaloTag (e) and HaloTag-actin (f) labeled with 0.1 nM and 0.01 nM JFX554-ligands, respectively. Single-plane images are acquired every 1 s for comparison with MYO7A movement. Kymograms are generated from the line scans between arrowheads. Most non-fused HaloTag molecules disappear after one frame (e, arrows) due to diffusion, except for a few molecules (e, open arrows). Most HaloTag-actin molecules stay in the same place and disappear due to photobleaching or transition to the dark state (f, arrows). Imaging conditions are similar to (b). Bars, 5 µm (cell images); 2 µm and 20 s (kymograms). Source data are provided as a Source Data file.