Figure 3. Single nucleosome imaging. (A) Generally, PA-GFP shows GFP fluorescence only after activation by a 405-nm laser (right). However, a small fraction of PA-GFP-H4 was spontaneously activated without laser activation (left) and was used for our single-nucleosome imaging. (B) A schematic representation of the oblique illumination microscopy (HILO system, ref. 37). We used Nikon laser TIRF microscope system Ti with Sapphire 488 nm laser (Coherent). Using a sheet light (blue), a limited area in the cell is illuminated. (C) Single-nucleosome image of a DM cell nucleus expressing PA-GFP-H4. PA-GFP-H4 observed as a bright dot using the oblique illumination microscopy system. For single-nucleosome tracking, the free software, u-track,⁴⁹ was used. The dots were fitted to an assumed Gaussian point spread function to determine the precise center of signals with higher resolution.³⁴ (D) Representative trajectories of fluorescently tagged single nucleosome.