We are starting to recruit PhD researchers for Oct 2021 start. Join a world-class, interdisciplinary and highly supportive research environment where you can learn about new cell biology projects and advanced microscopy techniques. Below, are the projects that are currently open to application.
- “Developing an ultra-fast and unbiased histopathology toolkit”
This is a 3.5-year fully-funded EPSRC DTP studentship based primarily in the Dept of Molecular Biology & Biotechnology, of the University of Sheffield. To read more or enquire, go to the FindAPhD page.
We are seeking enthusiastic candidates for an interdisciplinary PhD project developing a rapid histopathology tissue imaging tool.
Background: Histopathological examination of soft tissue biopsies is at the core of modern pathology and diagnostics. Despite being a manual process involving low-power microscopy and high error-rates, this remains the gold-standard for determining types of many cancers, muscle disease and connective tissue disorders. The utility and speed of these approaches are limited due to (a) the lack of sensitivity to use sub-cellular or molecular-scale features of the pathology in the diagnosis process and (b) human biases in recognising the pathological phenotypes. Whilst nanoscale imaging methods, commonly known as super-resolution microscopies, have existed for over 15 years now, they remain slow and difficult to adapt.
Overview: In this project, we will develop the protocol for rapid labelling of human biopsy samples with fluorescent nanodiamonds which will allow histopathological imaging of sub-cellular structures at nanometre-scale precision. Our recent work has demonstrated how the use of nanodiamonds can speed up super-resolution imaging by nearly 5-fold (DOI: 10.1101/2020.05.20.106716). We also recognise that the use of artificial intelligence (AI)-based image analysis and reconstruction algorithm is an opportunity to aid the recognition of cellular patterns associated with the disease phenotypes.
Objectives: This project will: (1) further speed-up nanodiamond-based super-resolution imaging of biopsies from scales of minutes to seconds, (2) characterise the spectral properties of spontaneous photoluminescence which underpins their ability to be rapidly localised, and (3) develop an AI-based real-time analysis to enhance the speed of sample scanning and assistive recognition of nanoscale pathology.
Approach: We will use antibody-conjugated nanodiamonds as molecular probes which target specific nanoscale structures of mitochondria. As a starting point, we will specifically image the cristae of mitochondria in muscle biopsy tissues taken from either diabetic or heart failure patients. A number of strategies will be used to further enhance this speed of imaging. These include adapting new camera technologies, enhanced sample illumination, extrinsic stimulation of the nanodiamonds with microwave and optimising the microenvironments of nanodiamonds and the tissue samples. A number of spectral analytical tools will be used to determine the spectral properties of spontaneous nanodiamond photoluminescence and match them to tissue types based on their intrinsic autofluorescence. Finally, we will adapt the framework of an existing AI-based image reconstruction platform to further accelerate the speed of imaging and to help identify shifts in mitochondrial structures that can indicate disease phenotypes.
The project will primarily be supervised by Dr Izzy Jayasinghe (https://appliedbiophotonics.org/). The student will have an opportunity to contribute to both existing and new collaborations with academic and industrial partners of the group. The second supervisor of the project will be Prof Ashley Cadby (https://ashleycadby.staff.shef.ac.uk/) who will advise on the adaptation of single molecule imaging technologies and software tools. The student will be a member of the Sheffield-based Imagine student cohort (http://www.imagine-imaginglife.com/) and will have the opportunity to present novel findings at conferences and public science communication platforms. The PhD training will focus on a broad range of cutting-edge research skills including (and not limited to) histology, super-resolution microscopy, advanced protein chemistry, advanced image analysis and computer programming.
Applications close on Mar 31, 2021.
2. “Novel molecular imaging tools for visualising the mechanisms driving vascular-immune cell diapedesis”
We are seeking enthusiastic candidates for an interdisciplinary PhD project to develop new imaging tools and novel insights into how immune cells ‘walk’ on the inner surface of blood vessels and migrate into the local tissue.
Overview: A key feature of the body’s adaptive immunity is the ability of immune cells to migrate in and out of the blood circulation. This PhD project will develop and apply a set of state-of-the-art imaging and cell culture tools to visualise the molecular machineries and mechanisms that underpin the close cell-to-cell interactions facilitating this process.
Background: The first step of the migration of leukocytes out of the blood vessels is the attachment to the vascular endothelium in order to gain access to the underlying interstitial space. At the sub-cellular level, this is triggered by cell-to-cell attachment and communication via a range of plasmalemmal receptors . A series of fast intracellular second-messenger signals and rapid remodelling of the membranes and cytoskeletons to form podosomes in both the endothelial cell and leukocyte ensue in a coordinated manner. This mutual motility of the endothelium and leukocytes – a process called ‘diapedesis’ drives the migration of the leukocyte. We will test the hypothesis that the strategic nanoscale organisation of these receptor/ signalling/ cytoskeletal complexes determine the shapes of the podosomes and the direction of diapedesis. Our approach will be to develop and apply a set of organoid and super-resolution imaging tools to gain visual insights into the molecular-scale orchestration of this process. By these molecular patterns and membrane topologies between healthy conditions as well as conditions that mimic local inflammation and local tumours.
Approach: We will establish a three-dimensional (3D) in vitro organoid system of vascular endothelial cells and local chemokine activation of immune cells. To optically map the complex topology and the receptor complexes of the leukocyte-endothelial interface, we will develop a new protocol of a super-resolution microscopy based on the recently-described “enhanced expansion microscopy (EExM)” method . With EExM, we will obtain a 3D molecular-scale fluorescent imprint of the membrane topologies, proteins and other cellular ultrastructures of the endothelial cells and migrating leukocytes onto an acrylamide hydrogel. Hydrating the hydrogel swells and expands this imprint, allowing us to perform super-resolution imaging to build up a spatially-accurate map of the molecules and membrane topologies involved. In order to validate and calibrate the molecular-scale measurements that we will make with EExM imaging, we will also develop a synthetic biomolecular calibration structure which can be embedded into the organoid sample. We will expose the apical and/or basolateral domains of the endothelial cultures to either pro-inflammatory cytokines  or tumour chemokines  to examine how the plasmalemmal receptor patterns and the podosome structure in disease conditions.
Supervision: The PhD student will be hosted within the laboratory of Dr Izzy Jayasinghe (https://appliedbiophotonics.org/) in The University of Sheffield. They will be given the opportunity to contribute to either existing or new collaborations with academic and/or industrial partners of the group. Co- supervisor of the project will be Dr Barbara Ciani (https://www.sheffield.ac.uk/chemistry/people/academic/barbara-ciani) who will advise on the development of biomolecular calibrants and training in the chemical biology facilities. The student will be a member of the Sheffield-based Imagine-student cohort (http://www.imagine-imaginglife.com/) and will have the opportunity to present novel findings at conferences and public science communication platforms. The PhD training will focus on developing a broad range of cutting-edge research skills including (and not limited to) 3D cell culture, super-resolution microscopy, advanced protein chemistry, advanced image analysis, spatial omics and computer programming. A good understanding of molecular or structural biology and protein chemistry will be desirable, although not essential.
[1.] Carman & Martinelli, Front Immunol. 2015; 6: 603.
[2.] Sheard et al., ACS Nano 2019 Feb 26; 13(2): 2143–2157.
[3.] Jose et al., Clin Transl Immunol. 2020; 9(5): e1131.
[4.] Yuki et al., Trends Immunol. 2020 Aug;41(8):652-664
Applications close on Mar 31, 2021.