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Histochem Cell Biol. 2012 Jan;137(1):1-10. doi: 10.1007/s00418-011-0880-5. Epub 2011 Nov 16.

Coordinate-based colocalization analysis of single-molecule localization microscopy data.

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1
Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, 97074 Würzburg, Germany.

Abstract

Colocalization of differently labeled biomolecules 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 resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization 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.

PMID:
22086768
DOI:
10.1007/s00418-011-0880-5
[Indexed for MEDLINE]
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