Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres

Szilárd Szikora; Tibor Novák; Tamás Gajdos; Miklós Erdélyi; József Mihály

doi:10.21769/BioProtoc.3654

Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres

Bio Protoc. 2020 Jun 20;10(12):e3654. doi: 10.21769/BioProtoc.3654.

Authors

Szilárd Szikora^{1

2}, Tibor Novák², Tamás Gajdos², Miklós Erdélyi², József Mihály^{1

2}

Affiliations

¹ Institute of Genetics, Biological Research Centre, Szeged, Hungary.
² Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary.

Abstract

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, ~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.

Keywords: Drosophila flight muscles; H-zone; IFM Analyzer; Quantitative localization microscopy; Sarcomere structure; Z-disk; dSTORM; rainSTORM.