Ems 575 x sputter coater

Coverslip cultures were fixed in 2.5% glutaraldehyde, 1% paraformaldehyde, 0.12 M sodium cacodylate buffer, pH 7.3, postfixed with 1% OsO4 in the same buffer, dehydrated in an ethanol series and critical point dried out of CO₂ in a Samdri-795 critical point dryer (Tousimis Research Corp, Rockville MD). The dried coverslip cultures were coated with 5nm of gold in an EMS 575-X sputter coater (Electron Microscopy Sciences, Hatfield PA) and imaged with a Hitachi S-3400 N1 scanning electron microscope (Hitachi High Technologies America, Inc., Pleasanton CA).

Optical imaging of fluorescent samples was performed with LSM 780 confocal laser scanning microscope (Carl Zeiss, USA). Once confocal images were obtained, culture dishes (MatTek glass bottom, P35G-1.5–14-CGR) were fixed in 2.5% glutaraldehyde, 1% paraformaldehyde, 0.1M sodium cacodylate buffer, pH 7.4, rinsed in cacodylate buffer, post fixed with 1% OsO4 in the same buffer, and dehydrated in an ethanol series. The coverslips were removed from the dish, dried using a Samdri-795 critical point dryer (Tousimis Research Corp, Rockville MD), coated with 5 nm gold in an EMS 575-X sputter coater (Electron Microscopy Sciences, Hatfield PA) and imaged with a Zeiss Crossbeam 540 (ZEISS, Jena Germany). Alignment of light and scanning electron microscopy images was done with the eC-CLEM plugin. Firstly, the LM image was aligned based on manually inserted landmarks. After this coarse alignment, a finer alignment was performed by registering the center of several (15 to 20) clearly identified labelled structures and their corresponding signals on the SEM micrographs.