Figure 1. Scheme of CLEM method.

(a) 100 nm ultrathin-cryosections are transferred from the cryo-ultramicrotome to the surface of a silicon wafer. This step is performed with a perfect loop and a drop of sucrose and methylcellulose. (b) Two pipettes are used to exchange solutions for immunolabelling so that sections remain wet. (c) The wafer is transferred upside down to a petri dish (glass bottom) with a drop of a 1:1 mixture of glycerin and PBS or imaging buffer for super-resolution, respectively. (d) A pipette is used to remove excess liquid from the sides of the wafer to bring the wafer nearly to the bottom of the petri dish (a very thin layer of solution is maintained). (e) Light microscopy is performed on inverted microscopes. (f) After imaging, a drop of PBS is applied from the side that allows the liquid to enter the space between wafer and the bottom. This gentle step will bring the wafer to the top of the drop and allows the wafer to be picked up without damaging or losing the section. (g) A drop of methylcellulose is added at 4 °C for 1 minute. (h) The wafer is transferred with forceps into an Eppendorf tube and immediately centrifuged at 14100×g for 90 sec. (i) The specimen is coated with Pt/C by unidirectional or rotary shadowing at an angle of 8°. (j) Imaging by SEM using secondary electrons. Design work produced by T. Gschwend, Multimedia and E-Learning Services, University of Zurich.