This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This version may be subject to change during the production process.

Edited by Wilhelm JustCryo-electron tomography (cryo-ET) provides high-resolution 3D views into cells pristinely preserved by vitrification. Recent technical advances such as direct electron detectors, the Volta phase plate and cryo-focused ion beam milling have dramatically pushed image quality and expanded the range of cryo-ET applications. Cryo-ET not only allows mapping the positions and interactions of macromolecules within their intact cellular context, but can also reveal their in situ structure at increasing resolution. Here, we review how recent work using cutting-edge cryo-ET technologies is starting to provide fresh views into different aspects of cellular biology at an unprecedented level of detail. We anticipate that these developments will soon make cryo-ET a fundamental technique in cell biology. Keywords: Cryo-EM; cryo-FIB; protein aggregationTransmission electron microscopy (TEM) is ideally suited to image biological structures at high resolution. However, water-rich biological samples need to be fixed to withstand the high vacuum of the electron microscope column. TEM preparation of cells and tissues most commonly involves chemical fixation, dehydration, resin embedding, and heavy metal staining. In contrast, cryo-electron microscopy (cryo-EM) relies on physical fixation by rapid freezing. If freezing is sufficiently quick the water molecules do not reorganize into crystals, but instead form amorphous ice. This vitrification process results in the exquisite preservation of biological material, which can be directly studied by cryo-EM [1]. Thus, cryo-EM allows high-resolution imaging of fully hydrated, unstained biological specimens.The most widespread application of cryo-EM is the study of purified macromolecules by the so-called 'single particle' approach [2]. Thousands of individual copies of the macromolecule of interest are embedded in a thin layer of vitreous ice and imaged in 2D. In principle, the macromolecules adopt random orientations within the ice, and therefore a sufficient number of images may provide all the angular views needed for high-resolution 3D reconstruction by averaging. Although such averaging procedures can only be applied to a set of near-identical objects, 3D imaging of unique biological objects such as cells can be performed by tomography. In tomography, 2D projections of the object of interest are recorded from different directions by tilting the specimen within the microscope. These projections are then computationally aligned for tilting imperfections and reconstructed into a 3D volume or 'tomogram' [3][4][5].Numerous cryo-electron tomography (cryo-ET) studies have showed the power of this technique to reveal the native architecture of labile cellular structures in situ [6][7][8][9][10][11].

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Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis of macromolecules in situ. Beyond such analyses of individual macromolecules, the study of their interactions with functionally related neighbors in crowded cellular habitats, i.e. ‘molecular sociology’, is of fundamental importance in biology. Here we present a NEighboring Molecule TOpology Clustering (NEMO-TOC) algorithm. We optimized this algorithm for the detection and profiling of polyribosomes, which play both constitutive and regulatory roles in gene expression. Our results suggest a model where polysomes are formed by connecting multiple nonstochastic blocks, in which translation is likely synchronized.

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