Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

Sandra Mayr,Fabian Hauser,Jaroslaw Jacak,Janett Göhring,Sujitha Puthukodan,Markus Axmann,Alessandro Esposito

doi:10.1371/journal.pcbi.1007902

Abstract

We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The in-depth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and -curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples. We demonstrated the biological importance of 2CALM, comparing the protein distributions of CD41 and CD62p on activated platelets in a 3D artificial clot. Additionally, using 2CALM, we quantified the impact of the inflammatory cytokine interleukin-1β on platelet activation in clots. The platform is applicable to any other cell type and biological system and can provide new insights into biological and medical applications.

Highlights

localisation microscopy (LM) has progressed immensely over the last decade [1,2,3,4,5], only a few of the approaches towards a comparative analysis of the resulting data have been achieved [6,7,8,9]
Companies and research facilities developed instruments for 3D/2D LM, but there is a lack of comparison methods for LM-images
Our system is capable of showing the difference on the level of single molecule clusters and provides information on differences between images on various scales

Summary

Introduction

LM has progressed immensely over the last decade [1,2,3,4,5], only a few of the approaches towards a comparative analysis of the resulting data have been achieved [6,7,8,9]. Analysis systems like SR-Tesseler [18] or ClusterVisu [19] based on Voronoi tessellation were developed These methods were well suited for visualization and rendering of localisation density distribution in a sample or colocalization between molecules primarily for 2D data. Clustering–the formation of micro- and nano-domains within plasma membranes–is a widely recognized feature that ensures hierarchical organisation of many proteins The functions of these clusters are diverse [20, 21] and impaired integrin clustering for example has been shown to be involved in thrombasthenia [22]. There is a general lack of methods that enable a comparative analysis of localisation microscopy data on protein distributions and clustering

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Discussion

Conclusion