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 50300 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.

Related Pub­li­ca­tions

  • 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), 778782. NCBI PubMed
  • 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), 815823. NCBI PubMed

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