Resource for the Visualization of Biological Complexity (RVBC)
Cryo-electron tomography
One of the primary core projects at the RVBC is development of cryo-electron tomography, a technique that provides high-resolution 3-D images of biological material in its native state - without structural disruption caused by chemical fixation, dehydration, or staining. Specimens are examined in the frozen-hydrated state, after use of rapid freezing methods that avoid formation of ice crystals that could damage the structure.
Thin films of suspensions of isolated organelles such as mitochondria, or small cells such as bacteria, are routinely prepared by plunge-freezing directly on EM grids. An FEI Vitrobot is available, which increases the control and reproducibility of plunge freezing. Preparation of tissue specimens is more elaborate: Small pieces of tissue are high-pressure frozen, which avoids formation of crystalline ice in depths up to about 100 μm. The major technical challenges are the subsequent ultramicrotomy and the mounting of the frozen-hydrated tissue sections so that they are suitably flat and stable during collection of a tomographic tilt series. The first electron tomographic analysis of frozen-hydrated tissue sections was published by the RVBC (Hsieh et al., 2002). Studies of the factors that contribute to section distortion and how to minimize them have been completed (Hsieh et al., 2006) and a totally novel approach to generate artifact-free frozen-hydrated sections has been pioneered using cryo-FIB milling of cell suspensions (Marko et al., 2006, 2007) and tissue (in progress).
Tomographic tilt series from frozen-hydrated specimens are recorded using either the IVEM (400 keV) or the F20 (200 keV, FEG). Both are equipped with CCD cameras and software for automated tilt-series acquisition using low-dose imaging. The IVEM also has an energy filter, which improves contrast in the tilt images by removing inelastically scattered electrons. To achieve resolution in the range of 3-4 nm, the cumulative electron dose on the specimen must be kept below a certain threshold, while at the same time maximizing the number of tilt images collected (McEwen et al., 2002). Recent experimentation has helped to optimize dose fractionation, i.e., determine the minimum dose per image possible for specimens of different thickness.
There are numerous computational challenges that must be met for high-resolution 3-D cryo-tomographic imaging. Recently completed and ongoing development projects include improving algorithms for aligning projections and computing the reconstructions, for determining the resolution of the reconstructions, and for visualizing the information contained in these noisy 3-D density maps. A motif search algorithm (Ramos) has developed that can detect the "signature" of known macromolecular complexes inside tomograms (Rath et al., 2003). A novel implementation of the Ramos algorithm was used to generate a partial map of the calcium release unit from multiple electron tomograms of frozen-hydrated triad junctions isolated from skeletal muscle (Renken et al., 2009).
Rendering from cryo-tomogram of isolated mitochondrion
References
Hsieh, C.-E., Marko, M., Leith, A., Mannella, C.A. and Frank, J. (2006) Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections. J. Struct. Biol. 153:1-13. Pub Med
Hsieh, C.-E., Marko, M., Frank, J., and Mannella, C.A. (2002). Electron tomographic analysis of frozen-hydrated tissue sections. J. Struct. Biol. 138:63-73. Pub Med
Marko, M., Hsieh, C., Moberlychan, W., Mannella, C., and Frank, J. (2006) Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy. J. Microsc. 222(Pt 1):42-7. Pub Med
Marko, M., Hsieh, C., Schalek, R., Frank, J. and Mannella, C.A. (2007). Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nature Methods 4(3): 215-217. Pub Med
McEwen, B. F., Marko, M., Hsieh, C.-E., and Mannella, C. (2002) Use of frozen-hydrated axonemes to assess imaging parameters and resolution limits in cryo-electron tomography. J. Struct. Biol. 138:47-57. Pub Med
Rath, B.K., Hegerl, R., Leith, A., Shaikh, T.R., Wagenknecht, T. and Frank, J. (2003) Fast 3D motif search of EM density maps using a locally normalized cross-correlation function. J. Struct. Biol. 144: 95-103. Pub Med
Renken, C., Hsieh, C., Marko, M., Rath, B., Leith, A., Wagenknecht, T., Frank, T., and Mannella, C.A. (2009) Structure of Frozen-hydrated Triad Junctions: A Case Study in Motif Searching Inside Tomograms. J. Struct. Biol. 165: 53-63. Pub Med