CLEM and confocal microscopy of SOs. HBMECs were either mock infected or adsorbed with reovirus T1L M1-P208S at an MOI of 1 PFU/cell and processed at 18 hpi for CLEM and confocal microscopy. (A) CLEM of infected HBMECs. The image on the left shows phase-contrast and fluorescence micrographs of infected cells stained with LysoTracker (red). The white dashed box marks the cell selected for EM; the black arrow indicates a lysosome close to a VI (white asterisks). Scale bar, 10 µm. Panels on the right show EM images of the selected cell. The arrow indicates the lysosome near a VI and the nucleus (N) observed in the previous image. This lysosome is an SO, as confirmed in the enlarged image of the third panel showing virions inside the SO (arrowheads). Scale bars, 500 nm. (B) Confocal immunofluorescence images of mock- and reovirus-infected HBMECs stained with phalloidin (green), LysoTracker (red), and an antiserum specific for σ3 (rabbit VU219). Arrows indicate lysosomes/SOs close to VIs, which are marked with asterisks. Scale bars, 10 µm. Live-cell imaging of lysosomes in uninfected and reovirus-infected cells are shown in Videos 2, 3, 4, and 5. (C and D) Tracking of lysosomes recruited to VIs in the same cell. Panels on the left and middle show single images of a live-cell microscopy video. The phase-contrast and fluorescence images show an infected cell stained with LysoTracker (red). Lysosomes adjacent to VIs are labeled by dark asterisks. The tracking data obtained with the TrackMate plugin of ImageJ-Fiji software are shown in the images on the right panel of C and right and middle panels of D. The specific trajectory of some lysosomes, most likely MCs (small colored circles), is marked by white arrows in D. Some lysosomes are first observed adjacent to VIs and then move to the plasma membrane (yellow dashed line, pm). Automatic tracks (marked with different colors) are shown in the right panel of D. A time-lapse recording is provided in Video 5. Scale bars, 10 µm.