Multi-Scale 3D Cryo-Correlative Microscopy for Vitrified Cells

Abstract

dimensional (3D) visualization of vitrified cells can uncover structures of subcellular 27 complexes without chemical fixation or staining. Here, we present a pipeline integrating three 28 imaging modalities to visualize the same specimen at cryogenic temperature at different scales: 29 cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron 30 microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark 31 revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast 32 cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone 33 Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be 34 applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-35 hydrated specimens in a variety of healthy and diseased conditions with and without treatments. 36 37 KEYWORDS 38 Airyscan microscopy; cryo correlative-light and electron microscopy, cryoCLEM; volume 39 cryo-focused ion bean scanning electron microscopy, cryoFIB-SEM; cryo-electron tomography, 40 cryoET; Hsp104 chaperone; protein aggregation 41 42 46 determination 1 . However, several limitations preclude its wider application, including the thickness 47 of some samples and difficulties in locating and identifying features of interest within them. To48 visualize molecular details in thicker samples by cryoET, such as regions in eukaryotic cells away 49 from the thin cell periphery, cryogenic focused ion beam scanning electron microscopy (cryoFIB-50 SEM) has been used to generate thin lamellae from vitrified cells 2-5 (i.e. thin layers through the 51 3 bulky cell) with a process called "ion beam milling", enabling many exciting biological observations 52 inside the cell 6 . However, technical challenges remain in ensuring that the milled lamellae contain 53 the features of interest. Correlative light and electron microscopy (CLEM) can overcome this 54 challenge by fluorescently labelling targets 7,8 , thereby guiding cryoFIB-SEM milling 9,10 and the 55 selection of optimal imaging areas for cryoET experiments, as well as aiding the interpretation of 56 observed features. However, even the smallest eukaryotic cells are typically several microns thick 57 and the desirable lamella thickness range is ~100-500 nm; thus, targeting nanoscale features 58 along the z direction remains extremely challenging. Here, as a proof-of-principle, we present a 59 pipeline to address this challenge by using high-resolution CryoAiryscan Confocal Microscopy 60 (CACM) to determine the z position of fluorescent targets within cells that were vitrified on electron 61 microscopy (EM) grids, followed by cryoFIB-SEM "mill and view" (MAV) imaging, which provides 62 a 3D view of whole cells with resolvable organelles as they are being milled to produce a lamella 63 containing the target of interest, and ending with visualization of molecular details of regions of 64 interest by cryoET. 65 66...

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