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.

Virus particle averaging and parameter fitting analysis. All analysis subsequent to dSTORM image reconstruction was performed using custom-written MATLAB routines (Mathworks). Briefly, the fluorescence image stacks obtained for mTurquoise and AF568 were independently averaged and converted into masks by thresholding. The masks obtained were applied to the dSTORM image. The protein labelled with AF568 was chosen such that it is located in a different structure than that labelled with AF647. When AF647 was used to label a protein present in the tegument, AF568 was used to label a protein in the envelope (glycoproteins). Inversely, if AF647 was used to label a protein from the envelope, AF568 was used to label a protein in the tegument. Virus particles present in the masked dSTORM image were then located by circular Hough transform 70 , allowing detection of circular structures and determination of the coordinates of their centre (see Supplementary Note 1).