To prepare intact cells or cytoskeletons for cryo-electron tomography it is necessary to grow cells on filmed EM grids. The samples must then be rapidly frozen in layer of medium thin enough to avoid ice crystal formation and also penetrable by the electron beam. This requires an apparatus which allows controlled blotting of the cells in a humid atmosphere to avoid drying before freezing.
For our experiments, we have used a semiautomatic instrument developed by Leica Microsystems in collaboration with Guenter Resch of the EM Facility, the Leica EM GP grid plunger. In this guillotine-like apparatus, the EM-grid carrying the specimen is mounted in forceps in a temperature– and humidity-controlled chamber. Subsequently, the major part of the liquid (medium, buffer) surrounding the specimen is blotted off with filter paper, to leave only a thin layer of 50–300 nm. Immediately after blotting, the specimen is plunged rapidly into a vessel containing a cryogen (liquefied ethane or propane). To avoid direct contact of the cells with the filter paper, the grids are blotted only on the backside and a blotting sensor ensures uniform results from one sample to the next. A high yield of well-frozen grids is particularly important when performing correlative light and electron microscopy. The frozen specimens can be stored in liquid nitrogen for long periods of time.
- Resch, G. P., Brandstetter, M., Pickl-Herk, A. M., Königsmaier, L., Wonesch, V. I., Urban. E. (2011). Immersion freezing of biological specimens: rationale, principles, and instrumentation. Cold Spring Harb Protoc. 2011(7), 778 — 782.
- Resch, G. P., Brandstetter, M., Wonesch, V.I., Urban, E. (2011). Immersion freezing of cell monolayers for cryo-electron tomography. Cold Spring Harb Protoc. 2011(7), 815 — 823.
- Leica EM GP on the Leica Microsystems Homepage