Cryo-electron tomography (CET) is the only method available for nanoscale study of the overall architecture fo cells in their native (frozen-hydrated) state. It is the only method capable of identifying and determining the structure and orientation of macromolecules in their cellular context. Specimens for CET are initially prepared by vitreous freezing;they remain fully hydrated, and no chemical fixatives or stains are used. However, because only specimens thinner than a few hundred nanometers can be effectively imaged in the electron microscope, use of CET has largely been limited to isolated cell organelles or small cells such as bacteria. Eukaryotic cells have been imaged only in special cases, and only at their peripheries. In order to extend CET to a broad range of cells and tissue, we were the first to use cryo-ultramicrotomy (mechanical sectioning) to prepare tissue sections sufficiently thin for CET. However, we have found sectioning of frozen material to be a difficult and poorly reproducible technique, fraught with unavoidable artifacts. Therefore, we have introduced an entirely new approach: cryo-focused-ion-beam (cryo-FIB) milling of vitreously frozen specimens for CET. Other laboratories have since confirmed our ground-breaking proof-of-concept experiments. Our goal in this proposal is to develop cryo-FIB milling into a practical, reliable tool for biological CET, and to enable the researcher to capture regions of interest in the specimen by correlative light and electron microscopy.

Public Health Relevance

We propose to provide a method that enables determination of the 3-D structure of any cell in its native, frozen, state by means of cryo-electron tomography (CET). Unlike traditional applications of CET, which are limited to very small cells, just the thin edges of cells, or bacteria and isolated cellular components, the new method will enable monolayer cell cultures, cell suspensions, and cells in tissue to be studied. This will have a broad and major impact on biomedical research.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097010-03
Application #
8518389
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Flicker, Paula F
Project Start
2011-08-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$496,724
Indirect Cost
$76,986
Name
Wadsworth Center
Department
Type
DUNS #
153695478
City
Menands
State
NY
Country
United States
Zip Code
12204
Hsieh, Chyongere; Schmelzer, Thomas; Kishchenko, Gregory et al. (2014) Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography. J Struct Biol 185:32-41
Sui, Haixin (2014) Inside out: tubulin cytomotive filaments versus microtubules. Structure 22:509-10