Zooming in and out: Exploring RNA Viral Infections with Multiscale Microscopic Methods

Figures

• 

  Figure 1. Schematic diagram showing the resolution capabilities of various imaging techniques in the research of RNA virus. Upper: resolution ranges of various imaging and structural methods. Lower: Schematic representations of the size scales of virus-infected cells, virus particles, and virus components (e.g., spike proteins). Different approaches can be utilized depending on the scale of the research object.

• 

  Figure 2. General workflow for microscopical research on RNA viruses. Samples are applied to grids and undergo a vitrification process (the most well-known procedure being plunge freezing), with subsequent imaging procedures depending on the sample. (a,b) For samples containing whole mammalian cells, serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) (a) and cryo-soft X-ray tomography (cryo-SXT) (b) can be used for volume imaging. (c) To achieve high-resolution imaging of regions of interest (ROI) within the cell (e.g., virus particles), a thin lamella of 80–250 nm can be prepared using FIB milling and imaged using cryo-ET. (d) Virions and VLPs can be imaged and structurally characterized using both cryo-ET or SPA. (e) For purified viral components (e.g., spikes), SPA is often the preferred method for structural analysis.

• 

  Figure 3. Cryo-SXT and cryo-FIB/SEM techniques are suitable for imaging and reconstructing intact virus-infected cells. (a) Three-dimensional reconstruction of whole-cell volumes of HCV replicon-bearing cells by cryo-SXT (scale bars, 1 µm) [9]. (i,ii) Tile-scanned projection (i) and overlaid with segmentation (ii) showing the area selected for SXT (white square); The enlarged view of segmented volume (iii) and tomographic slice (iv) from the boxed areas are also shown. Threshold-based isosurface segmentation of the surface boundaries identifies the different organelles present in the cells: abnormal mitochondria in red and purple, ER in yellow, modified ER in brown and nuclear envelope in pink. Purple arrows point to the abnormal mitochondria. (b) Characterization of SARS-CoV-2-infected Vero cells using cryo-FIB/SEM volume imaging [10]. Black arrows, SARS-CoV-2 virus particles; blue arrows, nuclear pores; dashed purple arrow, invagination path; Nuc nucleus; Cyto cytoplasm; V vesicles; T tunnels. (i) A representative cryo-FIB/SEM slice showing the invagination of cytoplasm into the nuclear space (scale bar, 1 µm). (ii) A representative cryo-FIB/SEM slice showing the distribution of…

• 

  Figure 4. Cryo-ET and cryo-EM for high-resolution characterization of viral structure in situ. (a) A cryo-correlative light and electron microscopy (CLEM) workflow targeting unlabelled MuV near fluorescently labelled mCherry-G3BP1 stress granules (SGs) [11]. (i) MuV-infected HeLa cells were seeded on an EM grid and imaged by phase contrast light microscope (scale bar, 200 µm). (ii) Infected cells with red fluorescent SGs (white arrowhead) are targeted for FIB-milling (scale bar, 10 µm). (iii) Lamella TEM map overlaid with cryo-confocal images confirming the presence of SGs post-milling and guiding cryo-ET data collection (scale bar, 2 µm) (Nu: nucleus). (b) Representative tomographic slice (left) and corresponding 3D segmentation (right) for in-cell MuV characterization (scale bar, 100 nm). Side and top views of MuV nucleocapsids are marked with cyan arrowheads and colored accordingly. The inset shows nucleocapsids have extended flexible densities (red arrowheads). Both the tomographic slice and segmentation are labelled with lipid droplets (LD), mitochondria (Mito), endoplasmic reticulum (ER), ribosomes (Ribo), and vesicles (V) [11]. (c) Subtomogram average of in-cell MuV nucleocapsids…