BackgroundConfocal 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.ResultsThe 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.ConclusionsVirtual 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1446-2) contains supplementary material, which is available to authorized users.

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.

Mitochondria and peroxisomes are dynamic signaling organelles that constantly undergo fission, driven by the large GTPase dynamin-related protein 1 (DRP1; encoded by DNM1L). Patients with de novo heterozygous missense mutations in DNM1L present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1) – a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we used human-derived fibroblasts from patients who present with EMPF1. In addition to elongated mitochondrial morphology and lack of fission, patient cells display lower coupling efficiency, increased proton leak and upregulation of glycolysis. Mitochondrial hyperfusion also results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential. Peroxisomes show a severely elongated morphology in patient cells, which is associated with reduced respiration when cells are reliant on fatty acid oxidation. Metabolomic analyses revealed impaired methionine cycle and synthesis of pyrimidine nucleotides. Our study provides insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1.

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.

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.