Structural analysis of herpes simplex virus by optical super-resolution imaging

Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

Particle averaging and model-based analysis of HSV-1. Nanometre-scale structural analysis by SMLM is a developing field. However, it requires high spatial resolution and high labelling specificity, and typically involves complex data analysis. This complexity is notably due to the need for assessing effects of the localization error and the linker size between the protein of interest and the fluorescent label 36, 37 . Here we present a structural analysis method taking these effects into account and apply it for the precise determination of the distribution of important tegument and envelope proteins in HSV-1. We acquired single-colour dSTORM images of purified viruses labelled with AF647. Here, a recombinant virus expressing mTurquoise-VP26 was used. We imaged a large number (450) of particles and data sets were analysed as shown in Fig. 2a (see also Supplementary Notes 1-3 and Supplementary Figs 1-3) . We ensured that only fully assembled virus particles, for example, containing capsid, tegument and envelope, were used for the analysis. For this, capsid-positive particles were selected by creating a mask from the mTurquoise-VP26 fluorescence image. A second mask was also created from an additional wide-field fluorescence image using AF568 as described in the Methods section.