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A novel method for quantified, superresolved, three-dimensional colocalisation of isotropic, fluorescent particles

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Abstract

Colocalisation, the overlap of subcellular structures labelled with different colours, is a key step to characterise cellular phenotypes. We have developed a novel bioimage informatics approach for quantifying colocalisation of round, blob-like structures in two-colour, highly resolved, three-dimensional fluorescence microscopy datasets. First, the algorithm identifies isotropic fluorescent particles, of relative brightness compared to their immediate neighbourhood, in three dimensions and for each colour. The centroids of these spots are then determined, and each object in one location of a colour image is checked for a corresponding object in the other colour image. Three-dimensional distance maps between the centroids of differently coloured spots then display where and how closely they colocalise, while histograms allow to analyse all colocalisation distances. We use the method to reveal sparse colocalisation of different human leukocyte antigen receptors in choriocarcinoma cells. It can also be applied to other isotropic subcellular structures such as vesicles, aggresomes and chloroplasts. The simple, robust and fast approach yields superresolved, object-based colocalisation maps and provides a first indication of protein–protein interactions of fluorescent, isotropic particles.

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Abbreviations

2D:

Two-dimensional

3D:

Three-dimensional

AU:

Arbitrary unit

DAPI:

4′,6-Diamidino-2-phenylindole

FRET:

Förster resonance energy transfer

GUI:

Graphical User Interface

HLA:

Human leukocyte antigen

LoG:

Laplace of Gaussian

MHC:

Major histocompatibility class

PMT:

Photomultiplier tube

PSF:

Point spread function

SNR:

Signal-to-noise ratio

LUT:

Look-up table

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Acknowledgments

We would like to thank the three anonymous reviewers for critical comments and pivotal suggestions, and J.A. Laissue, N. Kad and B. Amos for helpful discussions.

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Authors and Affiliations

  1. School of Engineering and Computing Sciences, University of Durham, Durham, DH1 3LE, UK

    Boguslaw Obara

  2. Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, OX1 3QG, UK

    Boguslaw Obara

  3. Oxford e-Research Centre, University of Oxford, Oxford, OX1 3QG, UK

    Boguslaw Obara

  4. School of Biological Sciences, University of Essex, Colchester, CO4 3SQ, UK

    Asma Jabeen, Nelson Fernandez & Pierre Philippe Laissue

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  1. Boguslaw Obara
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  2. Asma Jabeen
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  3. Nelson Fernandez
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  4. Pierre Philippe Laissue
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Correspondence to Boguslaw Obara or Pierre Philippe Laissue.

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Obara, B., Jabeen, A., Fernandez, N. et al. A novel method for quantified, superresolved, three-dimensional colocalisation of isotropic, fluorescent particles. Histochem Cell Biol 139, 391–402 (2013). https://doi.org/10.1007/s00418-012-1068-3

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