PhD position in advanced fluorescence microscopy applied to study stem cell metabolism
Successful organismal development and healthy tissue maintenance both rely on the activity of stem cells. During development, the earliest totipotent stem cells rapidly give rise to the blastocyst, from which pluripotent embryonic stem cells (ESCs) arise. These ESCs in turn commit to specific somatic cell lineages to eventually form tissues and organs of the body. For many years, stem cell metabolism was viewed as a by-product of cell fate status rather than an active regulatory mechanism. Yet accumulating evidence suggests that metabolism critically balances stem cell proliferation and differentiation. Elucidating how metabolic switches regulate cell fate decisions (so-called metabolic reprogramming) has since become a rapidly growing field of basic research with immediate relevance in regenerative medicine and diseases such as cancer.
This multidisciplinary PhD project concerns the application and further development of multidimensional fluorescence microscopy technologies including multiphoton microscopy and automated fluorescence lifetime imaging (FLIM) to explore how genome function, developmental potency and metabolism are connected in pluripotent stem cells. It will build on expertise in the Photonics Group and stem cell and developmental biology expertise in the Institute of Reproductive and Developmental Biology (IRDB) at Imperial College London. Specifically, we will investigate the principles and factors that maintain and fine-tune the pluripotent state towards differentiation, and how these modulations fundamentally link to metabolic switches, combining advanced fluorescence imaging techniques with metabolomics, genomics, and computational approaches. The project will be supervised by Paul French and Chris Dunsby in the Photonics Group and Véronique Azuara in the IRDB with further support from metabolomics and computational groups within the College.
Development and application of new technology for super-resolved microscopy
The recent Nobel prizes for super-resolved microscopy (SRM) underline the revolution tht has occured in optical imaging where the "diffraction limit" has been surpassed to enable features on a scale of 10's nm to be studied in detail, including in live samples. In the Photonics Group we have developed new SRM instrumentation for 3-D stimulated emission depletion (STED) microscopy, for single molecule localisation techniques such as PALM and STORM and for structured illumination microscopy (SIM) combined with fluorescence lifetime imaging (FLIM). We are seeking outstanding multidisciplinary research students who wish to further develop these technologies and apply them to challenges in cell biology, including the study of the cell cycle and the immunological synapse.
The ideal candidates would have a keen interest in the development and application of new methodologies for studying basic biology and would welcome the multidisciplinary nature of the project. They would have a first degree in physics, engineering or chemistry with strong practical skills and competence in programming for data acquisition and analysis. They would be required to acquire an advanced knowledge of optics and data analysis in the project and an enthusasm to learn the techiques associated with labelling proteins and cell biology techniques including culturing, transfecting and handling cells. This work would be supervised by Chris Dunsby, Mark Neil, Paul French and colleagues from Life Sciences and Medicine.
Please contact Paul French for further information.
High content analysis of 3-D cell cultures for drug discovery and basic research into disease mechanisms
Increasingly the use of conventional "2-D" cell cultures (typically monolayers of cells on glass or plastic substrates) are being found to be inadequate as models of biological systems for the purposes of fundamental biology research and drug discovery. This project, which would be undertaken in in the Photonics Group in collaboration with colleagues in biology, chemistry an d medicine, aims to develop automated assays of cell signalling processes in 3-D cell cultures, particularly tumour spheroids, where signalling processes are expected to be much closer to the in vivo context than for conventional 2-D cell cultures and therefore could provide more valuable information concerning cell biology and the response to drug candidates and also partially replace the need for animal testing. The project would build on earlier work developing FLIM FRET assays to read out protein interactions but would address new challenges in terms of the labelling, imaging and analysis of 3D cell cultures in a high throughput (HT) format and would specifically study the delivery and impact of novel drug candidates and inhibitory (therapeutic) antibodies into 3D cultures of mammalian cells.
The ideal candidate would have a keen interest in the development and application of new methodologies for studying basic biology and for drug discovery and would welcome the multidisciplinary nature of the project. They would have a first degree in physics, engineering or chemistry with strong practical skills and competence in programming for data acquisition and analysis. They would be required to acquire an advanced knowledge of optics and data analysis in the project as well as the chemistry associated with labelling proteins and cell biology techniques including culturing, transfecting and handling cells, especially in specialised 3D formats, during analysis.
Development of novel confocal/multiphoton endomicroscope systems for clinical diagnosis
At present, the diagnosis of many types of disease must be confirmed by histopathology before treatment can begin. This entails the physical removal of a small tissue specimen from the patient that is then processed, sliced, stained and analysed by an expert using an optical microscope. The collection of tissue is subject to sampling error, i.e. the diseased tissue may be missed, and the time required for processing and analysis can delay treatment. It would desirable for physicians to be able to make an immediate diagnosis during examination of the patient. Multiphoton microscopy can provide “optically sectioned” images of slices of tissue in vivo with sub-micron resolution, and clinical systems are commercially available for imaging skin. However, current multiphoton instrumentation cannot cope with patient movement artefacts and is unable to image curved areas of skin or to provide imaging during surgery or endoscopy. This project involves the development of novel multiphoton microscopy technology for imaging endogenous fluorescent molecules occurring in biological tissue in vivo. The first instrument is a novel lightweight hand-held multiphoton scanner, which would be able to compensate for patient motion (permitting longer acquisition times), richer spectroscopic readouts (including of fluorescence lifetime) and larger fields of view (to permit visualisation of whole lesions. This instrument will be applicable to image any external tissue or to tissues exposed during surgery. The second instrument is a disruptive technology concept invented and pioneered at Imperial for ultracompact multiphoton endoscopes of unprecedented size (<400 microns diameter) and flexibility, for use via fine needles directly in the organ of interest or via thin anatomical channels (down to breast ducts). It could thus provide sub-cellular imaging almost anywhere inside the body, including under the guidance of other imaging modalities (e.g. ultrasound, MRI). We are looking to recruit students to work on the development and application of these instruments alongside a team of Research Associates.
Optical projection tomography for 3-D preclinical imaging of disease models
THIS POSITION HAS NOW BEEN FILLED
There is currently tremendous excitement associated with new developments in optical imaging and particularly 3-D "mesoscopic" imaging of biological samples in the 100's μm to mm range. Such techniques can permit biological processes to be imaging in situ in live intact organisms, such as drosophila, nematodes and zebrafish. This PhD project concerns optical projection tomography (OPT), which is a mesoscopic imaging technique that is particularly suited for larger samples and has great potential for wide deployment of relatively low cost devices for real-world applications. The PhD research would be undertaken within the Photonics Group laboratories and also in collaboration with colleagues from the Department of Life Sciences and the Faculty of Medicine. The aim is to develop novel approaches to 3-D imaging of biological samples, including zebrafish for preclinical imaging of disease processes, e.g. associated with cancer, inflammation and bacterial infection. In particular, we are interested in developing new computational tools for compressive sensing and enhanced reconstruction of challenging 3-D tomographic data sets. Such tools would enhance our capabilities to study disease mechanisms and test new therapies. This project would require a student with a background in physics - including optics - with strong mathematical and programming skills and an enthusiasm for interdisciplinary research.
Receptor recycling and macrophage phagocytosis
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This is a joint project supervised by Chris Dunsby and Paul French in the Photonics Group and ouise Donelly and Peter Barnes in the NHLI. The role of macrophages is to clear and remove particles and pathogens and when this fails it may contribute to increased exacerbations and of progression chronic obstructive pulmonary disease (COPD). However, unlike macrophages in other parts of the body, under healthy homeostatic conditions, the lungs are not a serum-rich environment. This is important because most of the mechanisms into understanding the process of macrophage phagocytosis have focused upon opsonic uptake with little known about the mechanisms underlying non-opsonic phagocytosis. Phagocytosis is complex and requires engagement of cell surface receptors and activation of cytoskeletal rearrangements leading to particle engulfment and ultimate destruction inside the phagolysosome. This project will use human monocyte-derived macrophages to investigate the involvement and regulation of specific receptors and cytoskeletal proteins involved in the phagocytosis of different particles such as diesel particulates and pathogens including Haemophilus influenzae and Streptococcus pneumoniae. This will be investigated using flow cytometry and advanced automated fluorescence microscopy techniques in collaboration with the Photonics Group at Imperial College. Receptor trafficking following recognition of bacteria and particles will be examined using real-time microscopy to follow the fate of specific receptors. The role of the cytoskeleton will be investigated by transducing macrophages with lentiviral vectors that will express fluorescently labelled actin and tubulin to allow real time measurement of cytoskeletal rearrangements. To this end, we will use confocal and high content microscopy approaches including the automated optically sectioned (spinning disc) microscopy platform that is available in the Photonics Group providing multi-colour fluorescence intensity and lifetime imaging of fixed and live cells with bespoke image segmentation and quantification capabilities including FRET readouts of protein interactions. We will be able to visualize specific receptor localisation together with cytoskeletal components and will implement 3-channel imaging, using fluorescence lifetime and wavelength to separate labels, and will also explore using FLIM/FRET to read out ROS biosensors (such as HyPer) correlated with bacterial uptake and receptor internalization and use Duolink assays to investigate protein interactions. We will also explore the novel application of super-resolved nanoscopy techniques including SIM and STORM to investigate changes in cytoskeleton and receptor localisation and ultimately identify novel targets for improving macrophage function.
This multi-faceted project will entail training in both biological assays as well as the application and further development of advanced fluorescence techniques and is an exceptional opportunity to take advantage of the cross-disciplinary research environment at Imperial College. As such, it represented an opportunity for physical scientists or life-scientists wishing to broaden their expertise.
Video-rate volumetric light sheet microscopy for studying the interaction of induced pluripotent stem cell derived cardiomyocytes with mature cardiac tissue
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This project will develop and apply novel high-speed light sheet-based 3D microscopy technology developed in the Photonics Group in the Department of Physics. The project will involve modelling, development and modifications to sophisticated optical systems. It will also involve acquiring and handling large (TB) volumes of image data and developing computer algorithms to automatically analyse and quantify biologically relevant parameters.
The biological focus of the project is to understand how induced pluripotent stem cell (iPS) derived cardiomycotes cells interact and integrate with mature cardiac tissue. This is important because iPS derived cardiomycotes are an emerging therapy for the failing heart that have the potential to rejuvenate areas of heart tissue that have been damaged during heart attack. However, the integration of these new cells into the existing tissue and their subsequent function is hard to study using microscopy techniques that acquire images in only two dimensions. Changes in intracellular calcium concentration and trans-membrane voltage will be studied in 3D at video-rate as the wave-front of depolarization induced by electrical pacing spreads across and around the host and grafted tissue. Impulse propagation and induced cell contraction will be recorded in 3D to learn about the interaction of the iPS cells with their mature neighbours. Through these experiments, we will test if action potential duration dispersion and dys-synchrony of Ca2+ release (an index of excitation contraction (EC) coupling) is promoted by stem cell addition.
This is a fully funded PhD studentship, with joint funding from the Institute of Chemical Biology CDT and the British Heart Foundation Centre of Research Excellence. The initial MRes year will include lectures introducing physical scientists to cell biology.