FIG 7.

In situ hybridization, after short incubation times, of BSC-1 cells infected at a high MOI. (A) Cells were infected at an MOI of 20 for 15, 30, or 60 min with doubly labeled rcTLPs. Paraformaldehyde fixation was followed by overnight incubation with the pool of 44 Atto 565-labeled oligonucleotide probes and subsequent 3D imaging at 640-nm (left), 488-nm (middle), and 561-nm (right) excitation wavelengths, maximum-intensity projections of which are shown for a sample after a 60-min incubation. (B) Three fluorescently labeled particles in the red box in panel A, displaying the individual channels in the xz and yz planes through the center of each particle, as well as the maximum projection of the overlay of all three channels. White arrow, an uncoated DLP that colocalizes with ∼12 Atto 565-labeled oligonucleotides. Yellow arrow, an uncoated DLP with no colocalized oligonucleotide signal. Green arrow, an rcTLP. (C) Probability density of 640-nm channel amplitudes derived from image analysis as described in Materials and Methods. Detections with amplitudes of less than 900 were not considered a DLP, while detections above 4,000 were considered aggregates or multiple unresolvable DLPs. Red and blue arrows point roughly to the 640-nm amplitude of the particles in panel A highlighted by corresponding red and blue arrows. Particles such as these were excluded from analysis, as they likely represent aggregates or spatially unresolved particles. (D) Scatter plot of the number of Atto 565 probes colocalized with a given uncoated DLP after 15 (□), 30 (△), or 60 (○) min of infection at an MOI of 20. The particle highlighted in panels A and B is represented here as a red circle plotted within the 60-min data. (E) In images collected from mock-infected cells probed with the Atto 565-labeled oligonucleotide pool, 100 random “DLP” locations were generated, matching the average number of uncoated DLPs calculated from the data in Table 2. Of the 4,600 mock particles (black circles), 36 (red circles) colocalized with significant signal in the 560-nm channel as described in Materials and Methods. Quantification of the number of colocalized Atto 565 labeled probes was performed as described in Materials and Methods.