Development of oblique plane microscopy to accelerate our ability to understand and target complexity and heterogeneity in cancer
Developing new cancer therapies requires improved understanding of heterogeneity in cell behaviour. This requires determining single-cell responses, rather than the average responses of cell populations that fail to account for outlier cells that can be responsible for drug resistance. There is also increasing interest in more complex 3D cancer models, such as patient-derived organoids (PDO) that better recapitulate the complexity of the in vivo context compared to conventional high-throughput assays of homogenous 2D cell cultures. However, increasing the physiological complexity of cancer models makes them harder to image using optical microscopy techniques, and this makes screening many samples or conditions in high-throughput screening more challenging.
This PhD project is part of a Cancer Research UK funded Accelerator award between Imperial College London, the Francis Crick Institute, the Institute of Cancer Research (ICR), the University of Edinburgh and the Institute for Research in Biomedicine Barcelona. The project will develop, apply and utilise a new state-of-the-art automated light-sheet multiwell-plate oblique plane microscope (OPM) at the ICR to provide 3D imaging of 3D cell cultures and PDOs in 96 and 384-well formats. The PhD student will become expert in the use of the instrument, develop new image analysis pipelines and interpret these results in the context of the biology. The system will be used to provide quantitative single cell-resolved readouts and responses in fixed and live cell cancer models across a wide-range of conditions and provide functionality beyond the commercial state-of-the-art, e.g. providing faster 3D imaging of cell morphology, dynamics and migration in multiwell plate arrays. The project will enable studies of which cells within a heterogeneous cancer population are effectively killed by chemotherapy and which of the persisting cells are responsible for disease recurrence. It will also be used to explore the role of the tumour microenvironment for quiescent chemotherapy-resistant tumour cell sub-populations.
The ideal candidate would have a keen interest in the development and application of new tools and methodologies to study cancer biology and for drug discovery, and would welcome the multidisciplinary nature of the project. In particular they should have a strong physical- or computer-sciences background. A high level of computer literacy, including experience of computer programming is essential. The project is based at the ICR and will be co-supervised by Chris Bakal at the ICR and Chris Dunsby in the Photonics Group at Imperial.
3D polarization imaging for optical storage and quantitative biology
Data centres for cloud computing need to store large quantities of data in an archival format, where durability of the data is paramount. Examples of archive data include medical records, personal backups, insurance data or CCTV recordings. Currently archive data, also called “cold data”, is stored in magnetic tape which is only durable for a few years and data must thus be replicated periodically at huge cost. To address this issue, Microsoft Research is currently investigating the use of 5D high capacity polarisation multiplexed optical data storage in a collaborative research project called Project Silica. The 5D data storage technique uses a focused femtosecond laser to write data into the bulk of a glass block. At the focus of the laser a small ellipsoidal structure is formed with an orientation and size related to the input polarisation and intensity of the light respectively. Readout and recovery of such polarisation multiplexed data requires development of new advanced polarisation microscopes.
In addition to the field of optical storage, the question of measuring polarisation properties in three dimensions is very useful for biologists. Polarisation imaging modalities offer additional contrast mechanisms, allowing study of tissue birefringence or diattenuation. Collagen fibres, for example, exhibit both birefringence and diattenuation, the magnitude of which provides a measure of the local density and orientational uniformity, which in turn can give insight into the function and bio-mechanics of different structures. Furthermore, polarisation measurements can reveal the micro-structure and composition of tissues. Structural differences in, for example, elastin in skin can result from burns, photodamage or the development of skin cancer. 3D polarisation information can hence play a key role in tissue diagnosis and guided surgery, reducing the need for invasive biopsies or histological studies.
This fully funded three year EPSRC CASE project will be co-supervised by Prof. Mark Neil and Dr Matthew Foreman based at Imperial College London and Dr. James Clegg of Microsoft Research Cambridge. During the PhD project the student will develop models of the interaction of light with 3D polarisation sensitive samples and construct novel polarisation microscopy systems. These 3D microscopes will be used to measure 3D samples and demonstrate the recovery of polarisation data throughout a volume, in both optical data storage and biological contexts. The ideal candidate would have a first degree in physics or engineering with strong practical and modelling skills. They will build an advanced knowledge of optics through incorporation of theoretical and laboratory based elements and will have the opportunity to contribute to an exciting joint academic/industrial research project.
Accelerating our ability to understand and target complexity and heterogeneity in cancer through automated imaging of 3D cancer models including patient derived organoids
Drug resistance is a major challenge for cancer therapy, arising from heterogeneous cellular behaviours within tumours, where initially identical clonal cells can mutate and adapt to diverse microenvironments. This complexity is rarely addressed in standard assays that typically measure the average response of cell populations in highly artificial contexts and fail to account for outlier cells that may drive drug resistance. There is increasing interest in more complex 3D tissue models, such as patient-derived organoids (PDO) that better recapitulate the complexity of the in vivo context compared to conventional high throughput assays of homogenous 2D cell cultures. However, increasing the physiological complexity of cancer models makes them harder to image – limiting opportunities for high throughput (phenotypic) screening.
In a CRUK-funded Accelerator project, we aim to explore the trade-off between complexity of 3D cancer models and power of assays (in terms of single cell resolution and throughput) by developing and applying modular open source automated instrumentation optimised for 3D imaging of complex cell cultures with a range of optical properties. This automated 3D imaging instrumentation is intended to provide quantitative single cell-resolved readouts of drug-target engagement and responses in fixed and live cell cancer models. We will also explore cell culture and sample preparation (e.g. labelling, mounting, clearing) techniques to enable researchers to optimise 3D assays to address their specific cancer biology questions. Following instrument development at Imperial and the Crick, we will implement and apply these new capabilities, including with the University of Edinburgh, the ICR and the IRB Barcelona, to 3D cancer biology assays, e.g. to study heterogeneity in the response of cancer cells to chemotherapy - identifying which of the persisting cells are responsible for disease recurrence, to explore the role of the tumour microenvironment for quiescent resistant tumour cell sub-populations and to better understand side-effects of chemotherapy.
We seek to recruit two PhD students to be supported by 4-year CRUK-funded studentships (open to UK and EU candidates). The ideal candidates would have a keen interest in the development and application of new tools and methodologies to study cancer biology and for drug discovery and would welcome the multidisciplinary nature of the project - particularly the opportunity to gain practical expertise in new photonics technology and associated analysis tools. They would have a first degree in bioscience or physical sciences with strong practical and computational skills including competence in image data acquisition and analysis. They would be required to acquire an advanced knowledge of optics and advanced data analysis in the project, as well as the chemistry associated with labelling proteins and cell biology techniques including culturing, transfecting and handling 3D cell cultures. One PhD project would be undertaken with co-supervision by Erik Sahai at the Francis Crick Institute and Chris Dunsby & Paul French in the Photonics Group at Imperial. The second PhD project would be supervised by Iain McNeish in the Division of Cancer at Imperial, working closely with the Photonics Group.
Low-cost instrumentation for histopathology of kidney disease
Kidney disease is highly complex to diagnose, typically requiring light microscopy, immunohistology and electron microscopy (EM). However, EM is not available to much of the world’s population and it is challenging to maintain this expert service within the NHS. The goal of this fully-funded project is to replace EM with low-cost super-resolved microscopy for diagnosis of kidney diseases, including structural abnormalities of basement membrane and immune complex disease. We propose to develop a low-cost approach that could be accessible by clinicians in low and middle-income countries (LMIC) utilising our low-cost super resolving microscopy technique, “easySTORM” to image key proteins with clinically validated antibodies at a resolution sufficient to make a diagnosis. For this, we will develop low-cost instrumentation and practical protocols that will work with existing clinical biopsy samples.
This project will entail developing a practical instrument for histopathology based on our open source, low-cost, modular microscopy "openFrame" platform, implementing bright field and immunofluorescence microscopy as well as super-resolved imaging with automated slide-scanning. As well as being affordable to replicate, this instrument would be straightforward to maintain and would be tested in collaboration with colleagues from lower resources settings. It would be generally applicable in histopathology and, although this project will focus on developing protocols for diagnosing kidney disease, it could be extended to cancer and other diseases. As well as developing the hardware, the student would also develop and optimise specific protocols for sample preparation and imaging for the diagnosis of kidney disease, as well as suitable software tools for data acquisition and analysis, e.g. using MicroManager and ImageJ plug-ins.
The ideal candidate would have a keen interest in the development and application of new biophotonics technologies and would welcome the multidisciplinary nature of the project and the application to lower resoruced settings. They would have a first degree in physics or engineering with strong practical skills and competence in programming for image data acquisition and analysis. They would be required to acquire an advanced knowledge of optics and data analysis in the project and to learn sample preparation techniques for histopathology, including immunofluorescnce.
Please contact Paul French for further information. This fully-funded MRes+PhD studentship is available to UK and EU/EEA/Swiss applicants and would be part of the EPSRC CDT in Smart Medical Imaging, to which applications should be made before the deadline of 13 April 2020.
Biophotonics technology development for 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 and medicine at Imperial, the Francis Crick Institute, the ICR, the University of Edinburgh and the IRB Barcelona, aims to develop automated assays of cell signalling processes in 3-D cell cultures, particularly tumour spheroids and patient-derived organoids (PDO), 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 high content analysis (HCA) instrumentation for FLIM FRET assays to read out protein interactions but would address new challenges to improve the imaging performance and analysis of 3D cell cultures in a high throughput (HT) format through the development of new technology. The new 3D HCA instrumentation would specifically be applied to cancer, aiming to better understand and target complexity and heterogeneity in cancer through automated imaging of 3D cancer models including patient derived organoids.
The ideal candidate would have a keen interest in the development and application of new biophotonics technologies and would welcome the multidisciplinary nature of the project. They would have a first degree in physics or engineering with strong practical skills and competence in programming for image data acquisition and analysis. They would be required to acquire an advanced knowledge of optics and data analysis in the project and experience culturing, transfecting and handling cells, especially in specialised 3D formats, would be an advantage.
Light sheet fluorescence microscopy
Light sheet fluorescence microscopy (LSFM) is a high speed 3D fluorescence imaging method with the advantages of low photodamage to biological samples and low photobleaching of the fluorophores being imaged. However, conventional LSFM requires two microscope objectives placed at 90 degrees to one another to provide orthogonal illumination and detection. Oblique plane microscopy (OPM) is a technique developed in the Photonics Group at Imperial that uses a single high numerical aperture microscope objective to provide both the illumination light sheet and collection of fluorescence from the sample. In collaboration with the National Heart and Lung Institute, the current OPM system has been applied to image dynamic calcium events in isolated heart muscle cells at video volumetric imaging rates, which gives new insights into dynamic events that may trigger arrhythmias of the heart. It is also being applied in collaboration with the Institute of Cancer Research to achieve 3D imaging in 96 and 384 multi-well plates for higher throughput and time-lapse imaging of arrays of samples. Here, the aim is to study how different genes affect cancer cell morphology and the ability of cancer cells to change their shape over time, which is a factor that affecting metastasis in cancer. The aim of this project is to design, construct, test and apply the next generation of OPM microscope. The new system will provide higher spatial resolution and higher fluorescence collection efficiency than the existing system, and also be more compact. LSFM generates large image data volumes and so part of the project will be to develop custom code to provide automatic quantitative image analysis. The new system will be tested on a range of biological samples, including those from the collaborations mentioned above.
For more information see: https://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/oblique-plane-microscopy-opm/
or email Chris Dunsby
Next generation fibre laser pumped mid-infrared light sources
The mid-infrared (MIR) spectral region (~3-50 μm) is immensely important for applications in healthcare, manufacturing and defence. Key organic molecules exhibit strong and unique absorption of MIR light, including many atmospheric gases and complex proteins. Laser sources in the MIR are revolutionising the world we live in, for example, enabling the remote monitoring of pollution levels in our cities or accurately differentiating cancerous and healthy tissue in early-stage disease diagnosis. However, in comparison to the near-infrared (NIR), there are far fewer viable direct laser sources available in the MIR.
This project aims to use newly emerging nonlinear materials to convert established high-power NIR fibre laser technology to the MIR. Novel parametric conversion techniques will be developed, to demonstrate a set of unique laser sources that are compact, efficient and far outperform existing solutions. These sources will be deployed in target applications with academic and industrial collaborators around the world.
The student will be actively involved in the design, building and testing of high power fibre laser systems in the NIR, along with the corresponding wavelength conversion techniques necessary to generate MIR radiation. The project will involve working with collaborators at Imperial and internationally, providing the student with exposure to an exciting inter-disciplinary research environment.
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.
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.
Polarisation Imaging in Random Media
Development of quantitative techniques for measurement and monitoring of biological tissues is vital to improving healthcare and quality of life. Significant effort and resources have thus been invested to improve both the sensitivity and specificity of current bioimaging technology, with optical techniques at the fore. Predominantly, however, current methods are based on measurements of optical intensity or wavelength. Such measurements forego the additional information afforded by study of the degrees of freedom associated with the polarisation of light.
Polarization imaging modalities offer additional contrast mechanisms in biological imaging, such as quantification of collagen density through study of tissue birefringence or diattenuation. Furthermore polarisation measurements can reveal the micro-structure and composition of tissues, e.g. structural differences in elastin can result from burns, photodamage and/or the development of skin cancer. Although non-invasive in-vivo bioimaging methods are highly sought after, they can frequently be impeded by the need to image through relatively thick layers of highly scattering tissue, such as skin or breast tissue. Upon transmission of light through tissue the intensity and polarisation structure of the incident wave is modified, primarily due to scattering from e.g. cell nuclei or mitochondria, but also due to spatially varying birefringence and diattenuation from e.g. collagen networks.
This PhD project will focus on theoretically establishing novel techniques for polarisation imaging through disordered media for example by exploiting polarisation correlations and higher order statistical properties, machine learning and informatics. The student will develop analytic models to describe evolution of polarised light in random media and analyse a number of key problems including control of local polarisation in deep tissue, localisation and orientational measurements of buried fluorescent molecules and determination of structural properties of scattering tissues. The ideal candidate has a keen enthusiasm for theoretical optics and an interest in development of new applied methodologies for bioimaging. They would have a first degree in physics, engineering, or mathematics with strong analytical and programming skills.
Please contact Matthew Foreman for further information.
Optical projection tomography for 3-D preclinical imaging of disease models
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
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
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.