ORIGINAL PAPER Coordinate-based colocalization analysis of single-molecule localization microscopy data Sebastian Malkusch • Ulrike Endesfelder • Justine Mondry • Ma ´rton Gelle ´ri • Peter J. Verveer • Mike Heilemann Accepted: 19 October 2011 / Published online: 16 November 2011 Ó Springer-Verlag 2011 Abstract Colocalization of differently labeled biomole- cules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence reso- nance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-reso- lution methods offer the advantage of providing single- molecule coordinates that, rather than intensity informa- tion, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for locali- zation microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments. Keywords Colocalization  Super-resolution microscopy  Single-molecule fluorescence microscopy  Cellular structures Introduction Fluorescence microscopy is a valuable tool to unravel biomolecular interactions. By applying differently colored labels to biomolecules, interactions can be studied sys- tematically ranging from conventional colocalization down to the 2–10 nm length scale achieved by fluorescence resonance energy transfer (FRET) (Grecco and Verveer 2011). FRET is an excellent choice to study intermolecular interactions on a very short length scale, but requires that two biomolecules come together closely. On a longer length scale, spatial colocalization of the fluorescence signal in different color channels is a standard approach (Bolte and Cordelieres 2006; French et al. 2008; Zinchuk and Zinchuk 2008). However, colocalization of conven- tional fluorescence microscopy images suffers from a limited spatial resolution of about half the wavelength used and from chromatic errors (Ronneberger et al. 2008), which limits the extractable information and thus inter- pretation of the data. Here, super-resolution fluorescence microscopy techniques can help, as these methods can reach a spatial resolution down to tens of nanometers (Heilemann 2010; Schermelleh et al. 2010). Colocalization analysis has been applied for instance to stimulated-emis- sion depletion (STED) microscopy (McClatchey and Fehon 2009) and structured illumination microscopy (SIM) (Schermelleh et al. 2008) images. Localization-based super-resolution techniques are somehow different in this context as these are based on coordinates of single-mole- cules (Betzig et al. 2006; Heilemann et al. 2008; Rust et al. S. Malkusch and U. Endesfelder contributed equally. S. Malkusch  U. Endesfelder  M. Heilemann (&) Biotechnology and Biophysics, Julius-Maximilians-University Wu ¨rzburg, Am Hubland, 97074 Wu ¨rzburg, Germany e-mail: m.heilemann@uni-wuerzburg.de J. Mondry  M. Gelle ´ri  P. J. Verveer Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany 123 Histochem Cell Biol (2012) 137:1–10 DOI 10.1007/s00418-011-0880-5