We wish to study the physical mechanisms of energetic ion interaction with frozen biological specimens at temperatures in the range of 4-100K with the goal of improving specimen preparation for in situ cryoEM.
An ideal specimen preparation method was envisioned since the early days of TEM and dubbed the “most enjoyable dream” of electron microscopists by J. Dubochet. It comprises slicing a vitrified whole cell or tissue into a continuum of slices with appropriate thickness for high-resolution electron microscopy (2000 Å), and collecting these slices on a support suitable for imaging. In this ideal hypothetical method, no material would be lost or damaged at the interfaces of the slices, such that the entire volume can be imaged in series. No method at present is capable of this. Current sample preparation methods, such as FIB milling, can produce a single slice from the specimen, with thickness suitable for imaging in the TEM but also suffering from damage to the cut surfaces and several other problems. Being able to completely remove the damage layers and other artifacts would equate to reducing the total thickness of the lamella from 2000 A ̊ to 1400 A ̊. This, in turn, would improve the available signal by a factor of 1.3–1.4. The capability to produce such a slice, but completely undamaged, would be a major improvement on this method and our ability to image structures inside frozen cells.
Using experiments and simulations, we will seek to understand the mechanisms of damage to cryoEM specimens with ions and look to develop new instruments and methods to improve the thinning process.
References
J. Dubochet, M. Adrian, J.-J. Chang, J.-C. Homo, J. Lepault, A. W. McDowall and P. Schultz (1988)
Cryo-electron microscopy of vitrified specimens
Q. Rev. Biophys., 1988, 21, 129 —228.
C.J. Russo, J. Dickerson and K. Naydenova (2022)
Cryomicroscopy in situ: what is the smallest molecule that can be directly identified without labels in a cell
Faraday Discussions, 2022, DOI: 10.1039/d2fd00076h