Electron Cryomicroscopy of Fibrillar Collagens - Methods in Molecular Biology: Extracellular Matrix Protocols - page 100

Fig. 7. Diagram to show the fracturing of the mica ice wedge. Fracturing is achieved
by removal of the uppermost piece of mica using the microtome blade of the
Cressington instrument.

C generates mol-
ecules that are i) freeze-dried (those at the left-hand side of the mica, as shown); ii)
molecules adherent to the mica and submerged in frozen water or buffer; and iii) mol-
ecules that are at the surface of the frozen water or buffer. Molecules adherent to the
mica exhibit a progressive level of hydration (from left to right, as shown in the dia-
gram). Bracket shows zone of progressive hydration.

Fig. 8. Etching of the ice wedge by sublimation of water at -100 °

6. Freeze-etching of the ice wedge causes the ice level to recede, revealing fibrils in

a range of hydration states from containing bound water only, to being fully
hydrated ( Fig. 8 ).

7. Once the stage temperature has returned to -189 ° C shadowing and coating of the

sample can take place.

3.2.3. Shadowing and Coating
1.

Shadowing is achieved in the Cressington CFE-50C with a low-angle electron
beam gun loaded with a platinum/carbon rod and set at 11 to the sample. This is

then backed with carbon from an electron beam gun set at 90

°

° to the sample.

2. The platinum/carbon gun is set to evaporate 1-2 Å of platinum in the plane of the

mica substrate. The carbon gun is set to evaporate 5-8 nm of carbon for the film.