Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy

PLoS One. 2015 Jul 10;10(7):e0131756. doi: 10.1371/journal.pone.0131756. eCollection 2015.


Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation. Because the orientation varies with the length scale at which it is evaluated, we consider this scale as a separate informative dimension in the analysis. Additionally we introduce a data driven method for testing the statistical significance of the co-orientation and provide a method for visualizing the local co-orientation strength in images. We demonstrate our methods on simulated localization microscopy data of filamentous structures, as well as experimental images of similar structures acquired with localization microscopy in different color channels. We also show that in cultured primary HUVEC endothelial cells, filaments of the intermediate filament vimentin run close to and parallel with microtubuli. In contrast, no co-orientation was found between keratin and actin filaments. Co-orientation between vimentin and tubulin was also observed in an endothelial cell line, albeit to a lesser extent, but not in 3T3 fibroblasts. These data therefore suggest that microtubuli functionally interact with the vimentin network in a cell-type specific manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Algorithms
  • Animals
  • Cell Line
  • Cells, Cultured
  • Computational Biology / methods
  • Computer Simulation
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Human Umbilical Vein Endothelial Cells / metabolism*
  • Humans
  • Intermediate Filaments / metabolism*
  • Mice
  • Microscopy, Fluorescence
  • Models, Biological
  • Protein Binding
  • Tubulin / metabolism*
  • Vimentin / metabolism*


  • Tubulin
  • Vimentin

Grant support

R.P.J.N. and L.N. are supported by the Dutch Technology Foundation STW (, which is part of the Netherlands Organisation for Scientific Research (NWO) and which is partly funded by the Ministry of Economic Affairs, Agriculture and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.