Abstract
Cryo-tomography of intact, vitrified cells provides a three dimensional view of their structure and organization in a snapshot of the living state. Lacking heavy metal stains, tilt series images are typically produced by defocus phase contrast. Recently, a number of other methods have been introduced for 3D cryo-imaging. These include phase plate imaging, soft X-ray tomography, serial surface imaging using the focused ion beam-scanning electron microscope, and cryo-STEM tomography (CSTET). Here we explain the basis of the STEM setup and demonstrate the capabilities of CSTET to study unfixed, fully hydrated mammalian cells. Numerous cellular features are recognized in CSTET reconstructions, including membranes, vesicles, cytoskeleton, extracellular matrix, coated pits, and ribosomes. STEM signal acquisition configuration is more flexible than defocus phase contrast, and it imposes a much less severe spatial filter on the original images. Because low spatial frequency information is retained, the STEM tomographic reconstruction more faithfully represents the mass density distribution in the specimen.
Highlights
We explain the basis of the scanning transmission electron microscopy (STEM) setup and demonstrate the capabilities of cryo-STEM tomography (CSTET) to study unfixed, fully hydrated mammalian cells
Electron microscopy (EM) of biological systems is in the midst of major developments on multiple fronts
In special cases such as thin bacteria [3] or pico-eukaryotes [4], even entire cells can be studied by transmission electron microscope (TEM) tomography without sectioning, using standard defocus phase contrast, or more recently using newly available phase plates [5,6,7,8]
Summary
Electron microscopy (EM) of biological systems is in the midst of major developments on multiple fronts. Obviating the need for crystallization and easing requirements for rigorous purification, the way is open to study a far wider range of macromolecules and macromolecular complexes at high resolution. The second area of advance in EM encompasses methods for studying intact cellular structures and imaging macromolecular complexes embedded in their functional cellular contexts. In special cases such as thin bacteria [3] or pico-eukaryotes [4], even entire cells can be studied by transmission electron microscope (TEM) tomography without sectioning, using standard defocus phase contrast, or more recently using newly available phase plates [5,6,7,8]. Sub-tomogram averaging of structures detected within cells by cryo-tomography brings the power of single-particle reconstruction processing to a growing number of important in situ complexes and organelles [9]
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