Oneview 4kx4k camera

Purified human spermatozoa were collected and fixed with a freshly made fixative containing 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1M phosphate buffer with 4% sucrose (pH 7.2). After washing with 0.1M phosphate buffer with 4% sucrose, 0.1M glycine was added to quench the unbonded aldehyde group. The cells were dehydrated in graded series of ethanol on ice and embedded in LR White (Electron Microscopy Sciences, Hatfield, PA). The samples were polymerized under UV light (360nm) at −10°C for 48 h, followed by 12 h at RT. The 90 nm-thin sections were cut and mounted on Formvar-Carbon coated 200 mesh nickel grids. The grid was incubated with an anti-TFAM antibody (1:1000, Abcam, #ab119684) in PBS with 1% BSA, and 0.05% Tween 20, for 2 h at RT, then overnight at 4°C. Upon washing, the anti-mouse secondary antibody conjugated with 18 nm gold (1:15, Colloidal Gold AffiniPure Goat Anti-Mouse IgG (H+L, EM Grade, Jackson ImmunoReasearch Laboratories, Inc.,) were added in PBS with 1% BSA, and 0.05% Tween 20, and incubated for 1h. After washing, the grids were stained with uranyl acetate and lead citrate by standard methods, imaged with Talos120C transmission electron microscope (Thermo Fisher Scientific), and recorded using Gatan (4k x 4k) OneView Camera with software Digital Micrograph (Gatan Inc).

Negative staining was performed based on the following protocol^{22 (link)}. 3 μL of the sample was applied to a freshly glow-discharged carbon-coated grid. The grid was washed with two drops of deionized water and stained with two drops of 0.75% uranyl formate. After blotting off the excess liquid, the grid was vacuum aspirated and left to air dry for 20 min. The samples were viewed on an FEI Talos L120C TEM with Gatan 4k x 4k OneView camera at a magnification of 73,000x with a calibrated pixel size of 0.2 nm (Extended Data Fig.1D). The most promising sample judged by integrity and distribution of nucleosome particles was chosen for further cryo-EM experiments.