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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.

[Display omitted] • Applying SRRF nanoscopy under cryo condition. • Cryo-scanning transmission Electron tomography (CSTET) allows imaging of whole organelles and their interaction in 850 nm areas of intact and near-native cells. • Combining dual-axis CSTET with deconvolution results in visually isotropic resolution also in the axial direction. Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell. [ABSTRACT FROM AUTHOR]

Scanning transmission electron microscope (STEM) imaging has recently been applied to the cryo-tomography of thick biological specimens. As previously shown for plastic sections, STEM has a number of advantages for cryo-imaging compared to conventional wide-field TEM imaging. STEM is insensitive to phase coherence and is therefore suitable for much thicker specimens than TEM. Imaging in focus, with a long depth of field, also circumvents the complications of an oscillatory contrast transfer function and missing information at low spatial frequencies. Moreover the image signal represents a quantitative measurement of the electron scattering pixel by pixel, so that absolute intensities can be interpreted in terms of material properties in the specimen. Resolution, however, is undoubtedly compromised for thick samples, especially in the regime of multiple elastic scattering. In this work we address the specific issues that arise in cryo-tomography of thick biological specimens. We formulate an imaging model based on a Boltzmann transport equation, complemented by Monte Carlo simulations. Using these theoretical tools, we identify conditions for image acquisition that will be compatible with the basic presumption of tomographic reconstruction, i.e., that for a given composition the imaging signal varies monotonically with thickness. For optimal resolution, contrast, and signal strength, we propose to generalize the on-axis bright field detector to collect at angles well beyond the illumination cone. Our results justify the generation of 3D images for micron thicknesses and beyond. [ABSTRACT FROM AUTHOR]

Electron cryo-tomography using the scanning transmission modality (STEM) enables 3D reconstruction of unstained, vitrified specimens as thick as 1μm or more. Contrast is related to mass/thickness and atomic number, providing quantifiable chemical characterization and mass mapping of intact prokaryotic and eukaryotic cells. Energy dispersive X-ray spectroscopy by STEM provides a simple, on-the-spot chemical identification of the elemental composition in sub-cellular organic bodies or mineral deposits. This chapter provides basic background and practical information for performing cryo-STEM tomography on vitrified biological cells., (© 2019 Elsevier Inc. All rights reserved.)