Browsing by keyword "cisTEM"
Now showing items 1-2 of 2
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Cryo-TEM simulations of amorphous radiation-sensitive samples using multislice wave propagationImage simulation plays a central role in the development and practice of high-resolution electron microscopy, including transmission electron microscopy of frozen-hydrated specimens (cryo-EM). Simulating images with contrast that matches the contrast observed in experimental images remains challenging, especially for amorphous samples. Current state-of-the-art simulators apply post hoc scaling to approximate empirical solvent contrast, attenuated image intensity due to specimen thickness and amplitude contrast. This practice fails for images that require spatially variable scaling, e.g. simulations of a crowded or cellular environment. Modeling both the signal and the noise accurately is necessary to simulate images of biological specimens with contrast that is correct on an absolute scale. The 'frozen plasmon' method is introduced to explicitly model spatially variable inelastic scattering processes in cryo-EM specimens. This approach produces amplitude contrast that depends on the atomic composition of the specimen, reproduces the total inelastic mean free path as observed experimentally and allows for the incorporation of radiation damage in the simulation. These improvements are quantified using the matched filter concept to compare simulation and experiment. The frozen plasmon method, in combination with a new mathematical formulation for accurately sampling the tabulated atomic scattering potentials onto a Cartesian grid, is implemented in the open-source software package cisTEM.
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Locating Macromolecular Assemblies in Cells by 2D Template Matching with cisTEM [preprint]Over the last decade, single-particle electron cryo-microscopy has become one of the main techniques contributing to the growing library of high-resolution structures of macromolecules and their assemblies. For a full understanding of molecular mechanisms, however, it is important to place them into the broader context of a cell. Traditionally, this context can be visualized in 3D by electron cryo-tomography, and more recently, has also been studied by template matching of 2D images of cells and viruses. A current limitation of the latter approach is the high computational cost that limits the throughput and widespread adoption of this method. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software cisTEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated Mycoplasma pneumoniae cells and demonstrate that it can function as a versatile tool for in situ visual proteomics and in situ structure determination. We compare the results with 3D template matching of tomograms acquired on identical sample locations. We identify strengths and weaknesses of both techniques which offer complementary information about target localization and identity.
