Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural
organization is an absolute prerequisite to the functionality of these contractile
units. Despite the wealth of information collected, the exact spatial arrangement
of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed
a powerful nanoscopic approach to localize the sarcomeric protein components with
a resolution well below the diffraction limit. The ease of sample preparation and
the near crystalline structure of the Drosophila flight muscle sarcomeres make them
ideally suitable for single molecule localization microscopy and structure averaging.
Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins
with a quasi-molecular, similar to 5-10 nm localization precision. The protocol described
below provides an easy and reproducible method to prepare individual myofibrils for
dSTORM imaging. In addition, it includes an in-depth description of a custom made
and freely available software toolbox to process and quantitatively analyze the raw
localization data.
