Cryo-scanning transmission electron tomography of biological cells
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Three-dimensional cryogenic imaging of biological cells by electron microscopy Biological tissue is formed from individual cells, which present exquisitely complex and diverse morphologies. The study of their ultrastructure in three dimensions is almost synonymous with electron tomography.1,2 Early methods, such as serial sectioning for transmission electron microscopy (TEM), have been augmented by serial surface-imaging techniques in the scanning electron microscope, such as array tomography3,4 and iterative polishing by mechanical microtomy5 or focused ion beam milling.6,7 In the transmission electron microscope, tomography in tilt geometry is the preeminent approach to three-dimensional (3D) characterization. The 3D structure of a unique specimen, such as a biological cell, can be reconstructed from a series of projection images taken at different angles.8 TEM tomography has a number of differing implementations to suit the specimen preparation. Where cells or sections of tissue are embedded in plastic and stained with heavy metals such as uranium and osmium, the projection images are based on scattering contrast arising from the metal stain. Stain chemistry can be exploited to label specific structures or molecules within the ultrastructural context provided by 3D electron microscopy (EM).9,10 A new field of correlative
microscopy has emerged, combining fluorescence methods with EM for the best of both worlds.11–14 However, the steps of fixation and dehydration risk inducing changes in morphology; moreover, the stain distribution represents cellular features only indirectly. It is even possible to miss certain structures entirely if they are not effectively stained. Ultimate preservation of cells and tissues might be achieved if the sample could be immobilized instantly in place. This is the goal of cryogenic fixation, in which a fully hydrated biological specimen is cooled so rapidly (or under high pressure) that the water solidifies into an amorphous glass without crystallizing into ice.15–17 In the absence of heavy-metal staining, however, the light elements of biological matter scatter electrons too weakly to produce a conventional TEM image. Instead, contrast is generated by phase interference. Reconstruction from tilt tomography of phase-contrast images has emerged over the last dozen years as the state-of-the-art technique for the study of cellular architectures in unstained, cryogenically fixed material.18,19 The recent introduction of scintillator-free cameras (direct electron detectors) based on complementary metal oxide semiconductor sensors promises great improvements in in situ cellular tomography,20 as has already been demonstrated for purified macromolecules.21
Michael Elbaum, Department of Materials and Interfaces, Weizmann Institute of Science, Israel; [email protected] Sharon G. Wolf, Department of Chemical Research Support, Weizmann Institute of Science, Israel; [email protected] Lothar Houben, Department of Chemical Research Support, Weizmann Institute of Science, Israel; loth
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