Single-Molecule TIRF Microscopy of Halo-Tau

For performing single-molecule experiments, TIRF microscopy was performed using an Olympus excellence cell TIRF microscope equipped with 561-nm (200-mW) laser (Olympus, Tokyo, Japan) and a back-illuminated electron-multiplied charge-coupled device camera (C9100-13; Hamamatsu, Hamamatsu City, Japan). Fast single-molecule tracking used a digital complementary metal–oxide–semiconductor camera (ORCA-Flash4.0 V2 C11440-22CU; Hamamatsu). A 150× magnification objective with NA 1.45 (UAPON 150×/1.45; Olympus) was used for TIR illumination. The emitted light from the sample was filtered using a quad-band bandpass filter (FF01 446/523/600/677; Semrock, Rochester, NY). The microscope was enclosed in an incubation chamber maintained at 37°C and 5% CO₂ (Olympus-PeCon). Localization and trajectory reconstruction were carried out as previously described (Janning et al., 2014 (link)). The theoretical localization precision of Halo-tau was 16.7 ± 0.2 nm. For further analysis, a mask was placed over the cell processes, and trajectories outside of this mask were removed.