Browsing by Author "Theart, Rensu"

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    Virtual reality assisted fluorescence microscopy data visualisation and analysis for improved understanding of molecular structures implicated in neurodegenerative diseases
    (Stellenbosch : Stellenbosch University, 2020-03) Theart, Rensu; Niesler, T. R.; Loos, Benjamin; Stellenbosch University. Faculty of Engineering. Dept. of Electrical and Electronic Engineering.
    ENGLISH ABSTRACT: Confocal microscopy is one of the major imaging tools used in molecular life sciences. It delivers detailed three-dimensional data sets and is instrumental in biological analysis and research where structures of interest are labelled using fluorescent probes. Usually, this three-dimensional data is rendered as a projection onto a two-dimensional display. This can however lead to ambiguity in the visual interpretation of the structures of interest in the sample. Furthermore, analysis and region of interest (ROI) selection are also most commonly performed two-dimensionally. This may inadvertently lead to either the exclusion of relevant or the inclusion of irrelevant data points, consequently affecting the accuracy of the analysis. We present a virtual reality (VR) based system that allows firstly, precision region of interest selection and colocalisation analysis, secondly, the spatial visualisation of the correlation of colocalised fluorescence channels and, thirdly, an analysis tool to automatically determine the localisation and presence of mitochondrial fission, fusion and depolarisation. The VR system allows the three-dimensional reconstructed sample data set to be interrogated and analysed in a highly controlled and precise manner, using either fullyimmersive hand-tracking or a conventional handheld controller. We apply this system to the specific task of colocalisation analysis, an important tool in fluorescence microscopy. We evaluate our system interface by means of a set of user trials and show that, despite inaccuracies associated with the hand tracking, it is the most productive and intuitive interface compared to the handheld controller. Applying the VR system to biological sample analysis, we subsequently calculate several key colocalisation metrics using both two-dimensionally and three-dimensionally derived super-resolved structured illumination-based data sets. Using a neuronal injury model, we investigate the change in colocalisation between two proteins of interest, Tau and acetylated -tubulin, under control conditions as well as after 6 hours and again after 24 hours of neuronal injury. Applying the VR based system, we demonstrate the ability to perform precise ROI selections of 3D structures for subsequent colocalisation analysis. We demonstrate that performing colocalisation analysis in three dimensions enhances its sensitivity, leading to a greater number of statistically significant differences than could be established when using two-dimensionally based methods. Next, we propose a novel biological visual analysis method for the qualitative analysis of colocalisation. This method visualises the spatial distribution of the correlation between the underlying fluorescence channel intensities by using a colourmap. This method is evaluated using both synthetic data and biological fluorescence micrographs, demonstrating enhancement of the visualisation in a robust manner by indicating only truly colocalised regions. Mitochondrial fission, fusion and depolarisation events are important in cellular function and viability. However, the quantitative analysis linked to the localisation of each event in the three-dimensional context has not been accomplished. We extend the VR system to analyse fluorescence-based time-lapse sequences of mitochondria and propose a new method to automatically determine the location and quantity of the mitochondrial events. The detected mitochondrial event locations can then be superimposed on the fluorescence z-stacks. We apply this method both to control samples as well as cells that were treated with hydroxychloroquine sulphate (HCQ) and demonstrate how a subsequent quantitative description of the fission/fusion equilibrium as well as the extent of depolarisation can be determined. We conclude that virtual reality offers an attractive and powerful means to extend fluorescence-based microscopy sample navigation, visualisation and analysis. Three-dimensional VR-assisted ROI selection enable samples to be interrogated and assessed with greater precision, thereby exploiting the potential of fluorescence-based image analysis, such as colocalisation, in biomedical research. The automatic localisation and quantification of mitochondrial events can support research of mitochondrial function in healthy and diseased cells, where quantitative analysis of fission, fusion and depolarisation is of importance.