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Open Access Methodology article

Dual channel rank-based intensity weighting for quantitative co-localization of microscopy images

Vasanth R Singan1, Thouis R Jones23, Kathleen M Curran1 and Jeremy C Simpson4*

Author Affiliations

1 School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland

2 Imaging Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA

3 Institut Curie, Departement du Transfert, 75248 Paris, France

4 School of Biology and Environmental Science & Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland

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BMC Bioinformatics 2011, 12:407  doi:10.1186/1471-2105-12-407

Published: 21 October 2011

Abstract

Background

Accurate quantitative co-localization is a key parameter in the context of understanding the spatial co-ordination of molecules and therefore their function in cells. Existing co-localization algorithms consider either the presence of co-occurring pixels or correlations of intensity in regions of interest. Depending on the image source, and the algorithm selected, the co-localization coefficients determined can be highly variable, and often inaccurate. Furthermore, this choice of whether co-occurrence or correlation is the best approach for quantifying co-localization remains controversial.

Results

We have developed a novel algorithm to quantify co-localization that improves on and addresses the major shortcomings of existing co-localization measures. This algorithm uses a non-parametric ranking of pixel intensities in each channel, and the difference in ranks of co-localizing pixel positions in the two channels is used to weight the coefficient. This weighting is applied to co-occurring pixels thereby efficiently combining both co-occurrence and correlation. Tests with synthetic data sets show that the algorithm is sensitive to both co-occurrence and correlation at varying levels of intensity. Analysis of biological data sets demonstrate that this new algorithm offers high sensitivity, and that it is capable of detecting subtle changes in co-localization, exemplified by studies on a well characterized cargo protein that moves through the secretory pathway of cells.

Conclusions

This algorithm provides a novel way to efficiently combine co-occurrence and correlation components in biological images, thereby generating an accurate measure of co-localization. This approach of rank weighting of intensities also eliminates the need for manual thresholding of the image, which is often a cause of error in co-localization quantification. We envisage that this tool will facilitate the quantitative analysis of a wide range of biological data sets, including high resolution confocal images, live cell time-lapse recordings, and high-throughput screening data sets.

Keywords:
Quantitative co-localization; image analysis; non-parametric rank correlation; intensity weighting