Figure S2.

X-ray absorption data contrast in cells using different TXM objectives, related to Figure 2

(A) X-ray projection of a 16x16 μm FOV collected at the beamline B24 TXM using the 40 nm objective in a U2OS mammalian cell sample 1h after it has been exposed to infectious reovirus at MOI of 50. The zoomed view in square at the bottom left of the image is located outside the cell. The backing surface (Quantifoil™) can be identified by the regular pattern of circles (needed for blotting prior to vitrification).

(B) The diagonal dotted line defines the line used to generate (B), a line profile of pixel intensity in Fiji (Schindelin et al., 2012). Dips in the profile correspond to features of high X-ray absorbance such as membranes and organelles (corresponding vertical dotted lines have been added as examples). We expect reovirus particles to be present both in and outside the cell but given the high background it is be difficult to unambiguously identify.

(C and D) Closeups of marked areas showing contrast that could be attributed to reovirus particles.

(E) The associated line profile, clearly contains peaks that are 4-5 pixels apart; at 16nm per pixel that gives an object diameter of approximately 80 nm which matches the expected diameter of a reovirus particle. However, the ratio of pixel values is at best 1.1:1.3 even in this ‘ideal’ area (low background without any overlap with cellular content) making the unambiguous identification of individual virus impossible in this sample.

(F) X-ray projection of a 10x10 μm FOV collected at the B24 TXM using the 25 nm objective in the cytoplasm of a BSC-1 cell 16 h after it has been exposed to infectious reovirus at MOI of 50-100. The observed regular pattern of small dots is indicative of viral factories with each of these dots representing a single viral particle.

(G) A close up of the boxed area in (F), with a line drawn to generate (H), a line profile of pixel intensities. Full width half maxima of this plot give an average diameter of 60nm for these particles which appear to be an array of immature particles in the viral factory having yet to assemble an outer capsid before cell exit.