RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling

PLoS One. 2015 Sep 16;10(9):e0136233. doi: 10.1371/journal.pone.0136233. eCollection 2015.

Abstract

RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics*
  • Microscopy, Fluorescence / methods*
  • Nanotechnology / methods*
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics*

Substances

  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins

Grants and funding

This work was supported by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (to SJ), by the Max Planck Society, and by the DFG FOR 1086/2 (Grant SCHW 823/2-1) (MK, SPR Biosensor SR7000DC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.