Table 2.

Recommendations for detection of intact SARS-CoV-2 particles using electron microscopy in human autopsy tissues.

Criteria for ultrastructural identification
General considerations		It is sufficient if all of these criteria are met by a group of closely associated and similar particles within one individual cell, but individual particles of different cells should not be combined. Identification of cell types in autopsy tissues is challenging and often not possible, complicated also by pathological and virus-induced alterations that may mimick e.g. lamellar bodies of type 2 pneumocytes.
1	Shape	Round to oval.
2	Size	50-180 nm (mean = 87 ± 13 nm; without spikes), with smaller particles in re-embedded FFPE material (mean = 73 ± 7 nm, 58-108 nm). In the range also described for cell culture9 and autopsy lung.11
3	Membrane	At least partially visible around the particle.
4	Surface projections	Thin stalk and a globular component (in total about 20 nm long9), at least the globular component needs to be discernible with some distance from the biomembrane. The electron density is considerably lower than surface structures of e.g. coated vesicles and the particle surface usually is not entirely covered.9 Note that the visibility of surface projections may be heterogeneous within the sample and also depends on additives applied during tissue embedding such as en bloc treatment with tannic acid and uranyl acetate.9,25
5	Interior structure	Inhomogeneous granular (never empty or homogeneous at low electron density), ribonucleoprotein (RNP) profiles are round/aggregated or oval/longitudinal structures. Based on our findings, the RNP profile diameter is generally between 3.6-13 nm (mean = 7.2 nm ± 1.6 nm), as published.11,13
6	Number	Particles must be present at higher number and should often occur in groups within cells.3,12
7	Location	Extracellular: individual particles or small groups, sometimes attaching to outer surface of membranes. Intracellular: within small compartments with e.g. 1 particle up to very large compartments with dozens of particles, sometimes attaching to the inner surface of the membranes, but compartments with more than 1 particle should be identifiable as different structures such as swollen mitochondria may produce a “one-particle within a membrane compartment” appearance.

Recommendations for sampling and analysis
General considerations		Prioritize analysis of few, carefully selected samples with a high viral load. Controls are not required for virus identification, because EM allows a direct (label-free) proof of the respective particles.
1	Sampling	Analyse multiple blocks of the most strongly RT-qPCR-positive samples (see 3), facultatively at different levels to locate infection foci, even in apparently suboptimal samples, such as samples with a relatively long post mortem interval, or samples that have been frozen or paraffin embedded. Identify virus particles in the best specimen to get an internal “positive control”.
2	Correlation: IHC/ISH	Identify infection foci by using IHC or ISH and process corresponding FFPE regions via a punch biopsy or paraffin sections for EM as previously described.3,12 Serial paraffin sections may be processed to stain virus antigens and cell type markers and detect intact virus particles of the same cell.
3	Correlation: RT-qPCR	Try to roughly estimate the likelihood of finding virus particles based on RT-qPCR data as shown in our work with 0.006 or 500 expected particles per mm2 section area for low or high viral load, respectively.
4	Structural preservation/ image quality	Adjustment of fixation, embedding, sectioning and staining may be required for sufficient preservation of virus.7,43 Use adequate magnification to clearly resolve all relevant details of possible virus particles (0.2-1 nm pixel size) and adjust the respective EM settings correctly.43
5	Labelling techniques	Pre-embedding or post-embedding techniques should be used and interpreted with caution, as structural preservation usually is negatively affected. Morphological features of virus particles should still be identifiable and adequate controls should be used. Additional conventional EM for virus detection should be used.
6	Screening	Screen individual sections systematically at a medium resolution at which groups of viruses are easily detected (as can be tested by our large-scale datasets), and in some regions also at higher resolution to avoid missing of single virus particles present in the cells.
7	Pattern recognition	Learn the visual pattern of virus particles in tissue samples by using correctly identified virus particles (see large-scale repository datasets: http://www.nanotomy.org/OA/Krasemann2022eBioMedicine/index.html).
8	Figures	Ensure adequate image size, resolution, colour profile, brightness and contrast when publishing figures.