Resource for the Visualization of Biological Complexity (RVBC)
Cryo Specimen Preparation
Plunge-freezing
Two types of devices are available for vitreous freezing of suspensions of macromolecules, organelles, or small cells:
- A manually-operated double-blotting plunger constructed in-house.
- An FEI Vitrobot, which uses computer control to blot and plunge grids in a controlled environment.
In addition, a computer-controlled instrument is used for plunge-freezing in time-resolved experiments. The latter is described in the core research project on time-resolved cryo-EM.
High-pressure freezing
A Bal-Tec HPM-010 high-pressure freezer in Wadsworth Centers shared EM Core lab is available for RVBC use. The HPM-010 is used to prepare frozen-hydrated specimens for cryo-microtomy, and special specimen carriers are provided for that application. High-pressure frozen material can also be processed using a Bal-Tec FSU-010 freeze-substitution unit.
Cryo-ultramicrotomy
A Leica Ultracut UCT with an EM-FCS cryo kit is used for cutting frozen-hydrated sections. Special tools and accessories are included for that purpose. Several cryo diamond knives for trimming and microtomy, as well as an oscillating cryo diamond knife, are provided. The cryo-microtome chamber is shown below.
