Fig. 4

(A–D) Representative images of human osteosarcoma (HOS) cells expressing HaloTag-H2B fusion protein labeled with JF567-HaloTag ligand (7HTL; top) or JF570-HaloTag ligand (10HTL, bottom). (A) Pre-photooxidation fluorescence microscopy images. (B) Pre-photooxidation differential interference contrast (DIC) light microscopy images. (C) Post-photooxidation DIC light microscopy images. (D) TEM images post-photooxidation and OsO4 treatment of the entire field of view and regions of interest (ROIs) 1–4. ROIs 1 and 3 are from cells with low HaloTag-H2B expression and show low staining from DAB photooxidation and OsO4 treatment. ROIs 2 and 4 show increased electron density of the chromatin resulting from specific labeling of 7HTL or 10HTL and subsequent photooxidation of DAB and osmification; arrows highlight representative HaloTag-H2B-containing nucleosomes. Scale bars, 10 μm (A–D) and 200 nm (ROIs 1–4). (E) Representative electron tomogram slices in the nucleus of an HOS cell expressing HaloTag-H2B fusion proteins labeled with 10HTL, followed by photooxidization of DAB and osmification (cf. ROI 4). Arrows denote a cluster of nucleosomes in the volume. Scale bar, 200 nm. (F) 3D volumetric rendering extracted from the electron tomogram in (E). (G) Magnified view at the segmented chromatin denoted in (F). (H) Manual matching of stained nucleosomes in (G) with a simplified nucleosome structure consisting of a disc of 11 nm diameter and 5.5 nm height. These imaging experiments were duplicated with similar results.