1. Introduction – What is confocal microscopy and how does it work?
Confocal microscopy is a leading imaging tool used in molecular life sciences with which to render detailed high-resolution three-dimensional data sets. It is instrumental in biological analysis and research where structures of interest are labelled using fluorescent probes.
Typically, the three-dimensional data is rendered as a projection onto a two-dimensional display. However, this could lead to uncertainty in the visual interpretation of the sample’s structures of interest. Besides, analysis and region of interest (ROI) selection are also most commonly performed two-dimensionally which may inadvertently lead to either the exclusion of relevant or the inclusion of irrelevant data points. This, in turn, could affect the accuracy of the analysis.
2. Region of interest navigation and colocalisation analysis
In fluorescence microscopy, colocalisation refers to two probes, or colour channels, that co-distribute with each other. This can be used to determine whether two proteins or structures are associated with one another in the cell. Current tools are limited to calculating colocalisation on either a single micrograph of the z-stack or on a maximum intensity projection (MIP) of the z-stack. Therefore, current colocalisation analysis is limited to two dimensions, not allowing the isolation of defined regions of interest from the surrounding three-dimensional structure before calculating the colocalisation.
3. Objectives of the research
The overarching objective of this research is to develop a virtual reality-based method to visualise and analyse biological samples that can be used to improve the current understanding of cellular function. The specific research context chosen to apply the developed techniques is neurodegeneration and glioma cells. Therefore, to improve existing systems and methods currently employed for the analysis and visualisation of confocal image stacks we identified the following research objectives:
- To design, implement and evaluate a system that can spatially visualise colocalisation in a confocal microscopy sample using real-time interactive virtual reality, as well as calculating several commonly used colocalisation metrics in 3D.
- To implement different user interfaces appropriate for VR and to determine how intuitive and productive users are in using these interfaces.
- To design, implement and evaluate the various region of interest selection tools that allow 3D cell structures to be isolated from the whole sample and visualised by itself in virtual reality. Furthermore, to calculate colocalisation metrics within ROIs selected in this way and to compare these with the commonly used 2D colocalisation analysis.
- To design, implement and evaluate an improved means of spatially visualising the colocalisation in 3D and to compare this to existing methods by analysing different cellular structures.
- To design, implement, and evaluate an algorithm that automatically determines the location and status of mitochondrial events, specifically fission, fusion and depolarisation, and to visualise the result in 3D.
Dr Theart describes his research as “a virtual reality system that is capable of visualising and analysing three-dimensional cell samples with the hope of gaining a deeper understanding of how both healthy and sick cells function on a molecular level. We were mainly looking at neuronal cells that are found in the brain and considered different subcellular parts of the cells that are affected by neurodegenerative diseases such as Alzheimer’s disease in order to treat or prevent it.”
Virtual reality offers an attractive and powerful means of visualisation for microscopy data and, by using 3D analysis, samples can be interrogated and assessed with greater precision which means the potential of fluorescence-based image analysis in biomedical research can be better exploited. The research discussed above may serve as a basis for the development of further analysis tools with enhanced intracellular, 3D based precision analysis such as “the screening of drugs in the context of neurodegenerative diseases and cancer, where either mitochondrial dynamics are aimed to be maintained and preserved (for neurodegenerative diseases), or effectively disrupted (cancer)”.
Implicated in Neurodegenerative Disease – RP Theart