Super-resolution imaging of individual molecules of infectious HIV-1 before and after entry into lymphocytes. (a-c) Conventional total internal reflection fluorescence (TIRF) image (a), corresponding dSTORM image (b) and overlay of the TIRF (white) and dSTORM (red) images (c) of the HIV-1 matrix protein 20 minutes after synchronized entry into the lymphoid cell line MT-2. (d) Histogram of the localization precision values of molecular coordinates localized by dSTORM corresponding to the data set shown in a-c. Localization precision corresponds to one sigma of the Gaussian distribution of the point spread function that is fitted to individual molecules and is also affected by photons and noise level. Dashed line indicates mean. (e-h) Cluster analysis of the matrix protein in cell-free virions based on Ripley’s K-function converts the point distribution of molecular coordinates (e) into a cluster map with highly to less clustered regions colored red to blue (f). Cluster statistics such as number, size and associated molecules were extracted from thresholded images (g). By overlaying the TIRF image of GFP-Vpr (green) with the binary cluster map, the association of viral proteins with the reverse transcription complex after cell entry was quantified (h). Scale bars, 5 μm in panels in a-b; 1 μm in panel c and 2 μm in panels e-h. (i) Quantitative analysis of the diameter of the molecular clusters of capsid proteins in cell-free virions and 20 min post infection into MT-2 cells. Error bars represent the standard deviation of the mean from 26–173 clusters per sample from a representative from two experiments. *** = p < 0.001; ns = non-significant.