Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Abstract

DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous recombination repair or non-homologous end-joining, the question is not sufficiently answered, how a cell decides to apply a certain repair process at a certain damage site, since all different repair pathways could simultaneously occur in the same cell nucleus. One of the first processes after DSB induction is phosphorylation of the histone variant H2AX to γH2AX in the given surroundings of the damaged locus. Since the spatial organization of chromatin is not random, it may be conclusive that the spatial organization of γH2AX foci is also not random, and rather, contributes to accessibility of special repair proteins to the damaged site, and thus, to the following repair pathway at this given site. The aim of this article is to demonstrate a new approach to analyze repair foci by their topology in order to obtain a cell independent method of categorization. During the last decade, novel super-resolution fluorescence light microscopic techniques have enabled new insights into genome structure and spatial organization on the nano-scale in the order of 10 nm. One of these techniques is single molecule localization microscopy (SMLM) with which the spatial coordinates of single fluorescence molecules can precisely be determined and density and distance distributions can be calculated. This method is an appropriate tool to quantify complex changes of chromatin and to describe repair foci on the single molecule level. Based on the pointillist information obtained by SMLM from specifically labeled heterochromatin and γH2AX foci reflecting the chromatin morphology and repair foci topology, we have developed a new analytical methodology of foci or foci cluster characterization, respectively, by means of persistence homology. This method allows, for the first time, a cell independent comparison of two point distributions (here the point distributions of two γH2AX clusters) with each other of a selected ensample and to give a mathematical measure of their similarity. In order to demonstrate the feasibility of this approach, cells were irradiated by low LET (linear energy transfer) radiation with different doses and the heterochromatin and γH2AX foci were fluorescently labeled by antibodies for SMLM. By means of our new analysis method, we were able to show that the topology of clusters of γH2AX foci can be categorized depending on the distance to heterochromatin. This method opens up new possibilities to categorize spatial organization of point patterns by parameterization of topological similarity.

Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

“…Considering homeomorphisms and scale invariances within the detected γ-H2AX and repair protein clusters should therefore reflect topological patterns with high degrees of similarities. Recent results using persistent homology as an approach for SMLM data analysis and classification of γ-H2AX clusters after photon irradiation have revealed a dose-independent topological similarity of clusters depending on the neighborhood to heterochromatin [45]. The application of such approaches in future investigations will give further insights into complex damage processes and the relevance of chromatin architecture for repair process propagation.…”

“…After data analysis, artificial "pointillist" super-resolution images can be generated, in which the results of data analysis can additionally be encoded [40]. SMLM was applied to investigate the irradiation effects on chromatin nano-architecture [28,36,37] and protein organization at the nano scale [41][42][43], revealing systematic dose- [40,44] and locus-dependent [45] chromatin conformation changes. For multi-color localization microscopy of DNA damage markers along alpha tracks, we here applied molecular labelling with specific antibodies against γ-H2AX, MRE11, and 53BP1 in alpha-traversed cell nuclei and studied their distribution within γ-H2AX foci domains (super-foci) that demarcate the particle track.…”

Quantification of DNA damage induced repair focus formation via super-resolution dSTORM localization microscopy

“…These images can be further evaluated by using cluster analysis, and both the spatial distribution and the geometrical parameters of repair foci and even nanofoci can be determined. 32,33 Single molecule localization methods (SMLM), such as dSTORM, provide the highest lateral resolution among optical methods and open the way for imaging biological structures in the sub-20 nm regime. [34][35][36] SMLM determines the positions of individual molecules, which are used to create the final image.…”

Topological data analysis quantifies biological nano-structure from single molecule localization microscopy

“…Persistent homology is designed to reveal topological structure within pointillist datasets and has natural applications for the analysis of SMLM data, as also demonstrated in parallel work (31). Our novel framework moves beyond per cluster statistics such as area and density to reveal new information about the underlying topological features within segmented nano-structures.…”