Browsing by Author "Theart, Rensu P. "

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    Improved region of interest selection and colocalization analysis in three-dimensional fluorescence microscopy samples using virtual reality
    (Public Library of Science, 2018) Theart, Rensu P.; Loos, Ben; Powrie, Yigael S. L.; Niesler, Thomas R.
    Although modern fluorescence microscopy produces detailed three-dimensional (3D) datasets, colocalization analysis and region of interest (ROI) selection is most commonly performed two-dimensionally (2D) using maximum intensity projections (MIP). However, these 2D projections exclude much of the available data. Furthermore, 2D ROI selections cannot adequately select complex 3D structures which may inadvertently lead to either the exclusion of relevant or the inclusion of irrelevant data points, consequently affecting the accuracy of the colocalization analysis. Using a virtual reality (VR) enabled system, we demonstrate that 3D visualization, sample interrogation and analysis can be achieved in a highly controlled and precise manner. We calculate several key colocalization metrics using both 2D and 3D derived super-resolved structured illumination-based data sets. Using a neuronal injury model, we investigate the change in colocalization between Tau and acetylated α-tubulin at control conditions, after 6 hours and again after 24 hours. We demonstrate that performing colocalization analysis in 3D enhances its sensitivity, leading to a greater number of statistically significant differences than could be established when using 2D methods. Moreover, by carefully delimiting the 3D structures under analysis using the 3D VR system, we were able to reveal a time dependent loss in colocalization between the Tau and microtubule network as an early event in neuronal injury. This behavior could not be reliably detected using a 2D based projection. We conclude that, using 3D colocalization analysis, biologically relevant samples can be interrogated and assessed with greater precision, thereby better exploiting the potential of fluorescence-based image analysis in biomedical research.
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    Mitochondrial event localiser (MEL) to quantitativelydescribe fission, fusion and depolarisation in the three-dimensional space
    (Public Library of Science, 2020-12) Theart, Rensu P.; Kriel, Jurgen; Du Toit, Andre; Loos, Ben; Niesler, Thomas R.
    ENGLISH ABSTRACT: Mitochondrial fission and fusion play an important role not only in maintaining mitochondrial homeostasis but also in preserving overall cellular viability. However, quantitative analysis based on the three-dimensional localisation of these highly dynamic mitochondrial events in the cellular context has not yet been accomplished. Moreover, it remains largely uncertain where in the mitochondrial network depolarisation is most likely to occur. We present the mitochondrial event localiser (MEL), a method that allows high-throughput, automated and deterministic localisation and quantification of mitochondrial fission, fusion and depolarisation events in large three-dimensional microscopy time-lapse sequences. In addition, MEL calculates the number of mitochondrial structures as well as their combined and average volume for each image frame in the time-lapse sequence. The mitochondrial event locations can subsequently be visualised by superposition over the fluorescence micrograph z-stack. We apply MEL to both control samples as well as to cells before and after treatment with hydrogen peroxide (H2O2). An average of 9.3/7.2/2.3 fusion/fission/depolarisation events per cell were observed respectively for every 10 sec in the control cells. With peroxide treatment, the rate initially shifted toward fusion with and average of 15/6/3 events per cell, before returning to a new equilibrium not far from that of the control cells, with an average of 6.2/6.4/3.4 events per cell. These MEL results indicate that both pre-treatment and control cells maintain a fission/fusion equilibrium, and that depolarisation is higher in the post-treatment cells. When individually validating mitochondrial events detected with MEL, for a representative cell for the control and treated samples, the true-positive events were 47%/49%/14% respectively for fusion/fission/depolarisation events. We conclude that MEL is a viable method of quantitative mitochondrial event analysis.
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    Regression adjusted colocalisation colour mapping (RACC) : a novel biological visual analysis method for qualitative colocalisation analysis of 3D fluorescence micrographs
    (Public Library of Science, 2019-11-11) Theart, Rensu P.; Loos, Ben; Niesler, Thomas R.
    ENGLISH ABSTRACT: The qualitative analysis of colocalisation in fluorescence microscopy is of critical importance to the understanding of biological processes and cellular function. However, the degree of accuracy achieved may differ substantially when executing different yet commonly utilized colocalisation analyses. We propose a novel biological visual analysis method that determines the correlation within the fluorescence intensities and subsequently uses this correlation to assign a colourmap value to each voxel in a three-dimensional sample while also highlighting volumes with greater combined fluorescence intensity. This addresses the ambiguity and variability which can be introduced into the visualisation of the spatial distribution of correlation between two fluorescence channels when the colocalisation between these channels is not considered. Most currently employed and generally accepted methods of visualising colocalisation using a colourmap can be negatively affected by this ambiguity, for example by incorrectly indicating non-colocalised voxels as positively correlated. In this paper we evaluate the proposed method by applying it to both synthetic data and biological fluorescence micrographs and demonstrate how it can enhance the visualisation in a robust way by visualising only truly colocalised regions using a colourmap to indicate the qualitative measure of the correlation between the fluorescence intensities. This approach may substantially support fluorescence microscopy applications in which precise colocalisation analysis is of particular relevance.
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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 P. ; 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.
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    Virtual reality assisted microscopy data visualization and colocalization analysis
    (BioMed Central, 2017-02-15) Theart, Rensu P.; Loos, Ben; Niesler, Thomas R.
    Background: Confocal microscopes deliver detailed three-dimensional data and are instrumental in biological analysis and research. Usually, this three-dimensional data is rendered as a projection onto a two-dimensional display. We describe a system for rendering such data using a modern virtual reality (VR) headset. Sample manipulation is possible by fully-immersive hand-tracking and also by means of a conventional gamepad. We apply this system to the specific task of colocalization analysis, an important analysis tool in biological microscopy. We evaluate our system by means of a set of user trials. Results: The user trials show that, despite inaccuracies which still plague the hand tracking, this is the most productive and intuitive interface. The inaccuracies nevertheless lead to a perception among users that productivity is low, resulting in a subjective preference for the gamepad. Fully-immersive manipulation was shown to be particularly effective when defining a region of interest (ROI) for colocalization analysis. Conclusions: Virtual reality offers an attractive and powerful means of visualization for microscopy data. Fully immersive interfaces using hand tracking show the highest levels of intuitiveness and consequent productivity. However, current inaccuracies in hand tracking performance still lead to a disproportionately critical user perception.