NIHRDataInsights/HRCSData huggingface dataset:
Title: Molecular relativity: tracking single molecule movement relative to cell structures
Funder: Biotechnology and Biological Sciences Research Council

Abstract: In this project we aim to develop a method which can image both states of a photoswithable fluorophore simultaneously, giving data about the structure of the sample and the movement of individual molecules. We will then develop analysis techniques to track the movement of the structure and use it to correct the movement of individual molecules, so they are not influenced by sample motion or remodelling. This will allow us to accurately characterise the movement of molecules in motile cell structures. The performance of the software in correcting the positions of individual molecules will be characterised using simulations. Data will be collected using filopodia as a test system, and we will analyse the behaviour of the molecules to detect if there is any difference in the motion of molecules between fast moving and static filopodia, or between the tip and the main part of the filopodium. There is information present in the time domain of the high density channel. We will use this in two ways, once the movement of the structure has been eliminated from the time series. Firstly, frame sequences can be analysed using high density super-resolution techniques, which allow enhanced resolution to be achieved by fitting the positions of fluorophores in the high density data sequence. Secondly, the average movement of molecules within the sample will be analysed using spatio temporal image correlation spectroscopy. This will allow us to quantify how material is being transported in the sample at a micron lengthscale. This information will be compared with the information on single molecule movement to examine the difference between the characteristics of bulk transport and individual molecule movement. We will create an integrated software package and experimental protocol which will allow other users to carry out this type of experiment.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: An Aqueous Scanning Thermal Microscope for nanoscale thermal biology
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Scanning Thermal Microscopy (SThM) is a form of scanned probe microscopy that permits sample temperature to be measured and manipulated with miliKelvin resolution on the nanoscale. It is based around Atomic Force Microscopy probes that have a thermal sensor located at their tip. SThM has proved to be a powerful tool in investigating nanoscale thermal phenomena in the physical sciences. However, it is limited to operation in electrically insulating environments, such as vacuum, air or under oils. Attempting to use it under aqueous solutions results in immediate failure of the SThM probe through electrochemical corrosion, making the instrument unsuited to the vast majority of biological studies. In this research we propose to develop and evaluate a new SThM instrument (denoted as a-SThM) capable of operating in biologically benign aqueous solutions. The approach employed is based around electronic instrumentation that blocks electrochemical corrosion whilst still permitting electrical heating and interrogation of the SThM probe. As a consequence, commercially available SThM probes (or new a-SThM probes) can be employed in the system for biological studies. The new instrument will be of great interest to biologists as temperature is fundamental to all aspects of life, regulating many cellular functions from gene expression to cell metabolism. Despite this importance, much of our knowledge of thermal effects on cells is based on macroscopic measurements of temperature, resulting in the mechanism of temperature-induced changes at the subcellular level being poorly understood. Another obvious beneficiary will be research into nanoparticle-mediated thermal therapy, where localised hyperthermia offers great advantages in minimising damage to surrounding healthy tissues. However, a lack of suitable tools leaves determining the real temperature of individual nanoparticle heaters unsolved and identifying the appropriate dose for effective treatments extremely difficult.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Multiplex Bioorthogonal Labelling of Nucleic Acids: A Tool for Super-Resolution Imaging
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Molecular labelling reagents are important for marking cellular components to understand how they interact and regulate each other and how this is altered in disease. To date antibodies and protein tags are the reagents of choice for labelling proteins; in contrast fewer methods are available for labelling nucleic acids. This is particularly important for studying interactions within the nucleus using advanced imaging techniques such as super resolution microscopy where multiplex labelling reagents are required. The reaction between an azide and an alkyne, typically coined "click chemistry" is widely used for tagging DNA or RNA, separately. In this approach a modified nucleoside (with an azide or alkyne handle) is metabolically incorporated into the synthesised nucleic acid and this is subsequently labelled with a coupling partner, conjugated to a fluorophore. To complement this method we will develop a novel nucleoside analogue for labelling nucleic acids, within cells, that can be used combinatorially with azide/alkyne "click chemistry" to enable multiplex labelling of DNA and RNA, simultaneously. The approach is based on the inverse electron demand Diels-Alder addition between a strained-alkene and a tetrazine, a recently exploited bioorthogonal reaction that has fast kinetics, excellent orthogonality and biocompatibility. This reaction method will enable simultaneous multiplex labelling of DNA, RNA or other biological molecules. To demonstrate the utility of this approach we will analyse the distribution of an abundant nuclear protein SAF-A (scaffold attachment factor A) with DNA and RNA in mammalian cells, using super resolution microscopy. This proposal brings together chemists and molecular biologists to develop a new palette of tools for labelling nucleic acids and other biological molecules and showcases their use to investigate physiologically relevant molecular interactions in human cells.


Research activity codes: 1.3 CHEMICAL AND PHYSICAL SCIENCES
Health categories: GENERIC HEALTH RELEVANCE
Title: Functional Phenotype Flow Cytometer
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Historically cells have been catalogued in respect to their architecture by using fluorescent dyes to specifically label cellular and subcellular structures. Cell shape, organelle distribution and spatial localisation within the tissue have all been used to distinguish cell types. More recently, antibodies linked with fluorescent markers that can bind to individual molecules have been developed and used to differentiate cell types. However, such approaches are inaccurate, severely limited in their application to live cells and are difficult to use for classifying and separating cells based on functional phenotypic responses. Here we propose to use cell function to differentiate cell types. To our knowledge this approach is completely novel, we suspect largely because tractable tools have yet to be developed and demonstrated. Central to the functioning of living organisms, cellular activity is linked to characteristic intracellular processes. Research has generated many optical reporters for key intracellular mechanisms in living cells such as ion indicators, ATP/ADP sensors and cyclic AMP sensors and a great volume of knowledge has been gathered about the molecular stimuli that activate the corresponding intracellular activity in specific cell types. Given that the molecular stimuli and the associated intracellular process differ between cell types, here we will develop a flexible opto-microfluidic device that sorts cells based on their functional characteristics. By analysing in real time the cell responses to specific stimuli, we will use the proposed device to isolate cells from a mixture that specifically respond to one or more stimuli and thus sort cell based on their functional phenotype. Proving the versatility of the device we will also employ the platform for real time analysis of cell function providing a high throughput platform for studying interactions of the cells with specific stimuli.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Ultra-Sensitive and Ultra-Fast Absorption Spectrometer for Micro-Droplet-based Enzyme Evolution Experiments
Funder: Biotechnology and Biological Sciences Research Council

Abstract: High-throughput, ultrafast and ultrasensitive absorption measurements in micro-droplets can be achieved by integrating whispering-gallery mode optical sensors with state-of-the-art micro-droplet fluidics. Our idea is to monitor the optical losses as the light propagates around the circumference of a micro-drop by total internal reflection, allowing us to measure absorption and therefore study chemical changes within the droplet. The speed, throughput and sensitivity of this droplet-based absorption sensor can surpass currently available droplet-based spectroscopies: the sensitivity as compared to a single pass droplet absorption spectrometer is greatly enhanced, approximately by three orders of magnitude due to the more than ~1000 roundtrips of the light inside the drop. By combining this novel technology with synthetic biology approaches for expressing diverse DNA templates we will build a new tool for high throughput investigation of enzyme function, mutational permutations of enzyme function and screening meta R/DNA templates for discovery of novel enzyme functions.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: New Technology for High-Performance and High-Capacity Bio-separations: Protein Purification using "Hydrophobic Water" Solvent Systems
Funder: Biotechnology and Biological Sciences Research Council

Abstract: New Technology for High-Performance and High-Capacity Bio-separations: Protein Purification using "Hydrophobic Water" Solvent Systems in Countercurrent Chromatography Apparatus Technical Summary This project makes use of newly discovered ionic liquids and microemulsion solvent systems derived from ionic liquids, for use in modern countercurrent chromatographic (CCC) equipment. CCC separations are based on the partitioning of solutes between two immiscible solvents. It is proposed that a combination of (1) ionic liquids, (2) modern high performance countercurrent chromatography (HPCCC) and centrifugal partition chromatography (CPC) and (3) co-solvent free, high water content microemulsions be used to separate peptides and proteins. The microemulsion solvent systems that would be used contain a surface active, hydrophobic ionic liquid / water / hexane mixture. These have water as a mobile phase and a high-water content microemulsion as a stationary phase in the CCC machine. This will enable a range of high performance, high capacity and environmentally friendly separations of peptides, proteins and other biomolecules to be carried out. Since the mobile or carrier phase in these separations is usually water, this makes the separation process cost effective, environmentally benign, robust and tolerant of suspended solid matter in the samples (which would normally destroy a HPLC column). In addition, the whole process can be scaled up and automated to minimize protein production costs.


Research activity codes: 1.4 METHODOLOGIES AND MEASUREMENTS
Health categories: GENERIC HEALTH RELEVANCE
Title: Mesolab: A Centre for Optical Mesoscopy for Biomedical Research at the University of Strathclyde
Funder: Biotechnology and Biological Sciences Research Council

Abstract: It is a great advantage in biomedical research if the field of view can be extended to cover the entire object, e.g. an embryo or an organ, without sacrificing sub-cellular detail in the image. In a conventional microscope any attempt to view large fields reduces the spatial resolution. Not only is detail lost in the image, but depth discrimination (required for confocal function) is strongly compromised. What is needed is a microscope lens with a low magnification (e.g. 4x) and yet a high numerical aperture (approaching 0.5). The Mesolens was designed by our Project Partner Mesolens Ltd to meet this need. We propose a Mesolab facility at the University of Strathclyde equipped with two Mesolens systems. The first will use a high-end camera capable of high-spatial resolution recording at >96 MP and a smaller-chip, fast, high sensitivity sCMOS camera, for wide-field imaging and digital lightsheet scanning mesoscopy. The second system will also have modest epi-fluorescence capability but will be used primarily for laser scanning confocal (already demonstrated), multi-photon and fluorescence lifetime mesoscopy. We will use the optical mesoscopes to answer a host of key technical biomedical questions, including the following: can every cell in a mouse embryo be imaged using optical mesoscopy and what are the best preparative methods? Will this pinpoint the cellular basis of genetic defects? How deeply can these optics penetrate into living tissue? How much can the duration of fluorescence studies be increased because of the optical mesoscope's improved light collection? Can cell migration inside tumours be followed by using bioluminescent markers, as we suspect? To our knowledge, no similar technology is being developed anywhere in the world. We have a head-start in our unique achievement of adapting a Mesolens for laser scanning imaging and we have the right expertise in optical physics and software design to drive this project forward.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: MICA: Nanoscopy Oxford (NanO): Novel Super-Resolution Imaging Applied to Biomedical Sciences
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The past decade has seen a major revolution in optical microscopy, with new methods surmounting the conventional diffraction limit in spatial resolution. Super-resolution microscopy (SRM) techniques such as stimulated emission depletion (STED), REversible Saturable/switchable Optical Linear (Fluorescence) Transition (RESOLFT), (Saturated) Structured Illumination Microscopy ((S)SIM) and (fluorescence) PhotoActivation and Localization and (direct) Stochastic Optical Reconstruction microscopy (PALM/(D)STORM) permit imaging of living cells with increasing resolution and are opening a broad frontier in the study of cellular functionality. Each of these methods has distinct advantages and disadvantages for imaging different types of specimen. The principle aim of this proposal is to make all developments in SRM available to the biomedical community in Oxford to enable unique insights in cellular research. We already have a well-established infrastructure to support advanced optical imaging within the University with the Micron Advanced Bioimaging Unit and planned facilities in the WIMM and at WTCHG. There are two SIM platforms in Micron but we will establish STED and RESOLFT imaging at the WIMM and provide access to a home-built PALM/STORM system in Micron. SRM techniques have enormous potential, yet their utility is still restricted by a number of inherent limitations. A second aim is to grasp this opportunity to unite biologists with physicists, engineers and chemists to drive further developments in the technology of SRM imaging. We will develop: 1. STED and RESOLFT approaches in Oxford. 2. SSIM with fast image acquisition at a spatial resolution of 50nm for 3D imaging. 3. adaptive optics for the correction of aberrations encountered when performing SRM at depth within tissue. 4. live PALM/STORM and FRET. 5. new image analysis and control algorithms for the super-resolved SRM data. 6. fluorescent labels for live-cell SRM.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Super Resolution Imaging for Cell Biology and Neuroscience at UCL
Funder: Biotechnology and Biological Sciences Research Council

Abstract: We aim to develop a super-resolution fluorescence imaging (SRFI) facility that will link MRC scientists in the Cell Biology and Prion Units, as well as the MRC/UCL Centre in Molecular Medical Virology, with members of UCL's Faculties of Life Sciences, Medicine and Brain Sciences, the London Centre for Nanotechnology, UCL physicists and chemists, computer scientists, image-based bio-informaticians and the National Physics Laboratory, Teddington (NPL). The facility will house commercial SRFI instruments for use on a range of projects with particular emphasis on cell biology, neurobiology and infectious disease. These instruments will provide state of the art bench-marks against which UCL and NPL researchers will develop new instruments, optimised for biomedical research. We are requesting funds to:- 1. Purchase a Zeiss Elyra PS1 that will provide PALM/STORM and SIM capability, providing a multi-user platform through which UCL (Lowe, Kittler) and NPL (Knight) PALM/STORM systems and the NPL SIM system can be developed, compared and optimised. We will use UCL funds to buy a second commercial instrument to offer a broad range of imaging options. 2. Support two postdoctoral RAs to train/work at the interface of biology, imaging and instrument development under the guidance of the facility's Management Board. There will be additional capacity-building opportunities linked to the facility for UCL students and postdoctoral fellows. 3. Support development, including cloning the NPL and Lowe lab. instruments for use by UCL researchers. Significantly, the software for these instruments will be open source, allowing better assessment and control over data processing/analysis. The facility will enable research into fundamental questions in biomedical research, providing new insights to disease mechanisms and opportunities for therapeutic development. The facility and its Management Board will also provide a focus to coordinate imaging developments at UCL.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Next generation live super-resolution microscopy: development and application at the Cambridge Advanced Imaging Centre
Funder: Biotechnology and Biological Sciences Research Council

Abstract: We will custom-develop 2 separate instruments for 1) far-field individual molecule localization with Total Internal Reflection Fluorescence (TIRF) and 2) gated Stimulated Emission Depletion microscopy (g-STED) & Reversibly Switchable Optical Fluorescence Transition using reversible photoswitching GFP (rsGFP-RESOLFT). Instrument 1) will implement the Double Helix Point Spread Function detection (DH-PSF) technique to axially encode the position of single emitters from direct Stochastic Optical Reconstruction Microscopy (dSTORM) or PhotoActivation Localization Microscopy (PALM). DH-PSF can typically attain a resolution of 10-15 nm laterally and 20-25 nm axially. Instrument 2) will readily achieve a planar resolution of 70 nm, and 300 nm axially in fixed tissue in g-STED operation. This instrument will also serve as development platform for rsGFP-RESOLFT. In collaborative research with the inventors of RESOLFT, we will implement 1) parallel STED beam excitation and readout using microlens arrays and EMCCD detectors to shorten overall acquisition time; 2) improve z- resolution through the use of two individual phase plates to generate a 3-dimensional RESOLFT 'doughnut'. Building on our expertise in Fluorescence Light Sheet Microscopy (FLSM), we will combine FLSM with PALM/dSTORM and DH-PSF detection to enable super-resolved imaging of large cells (>5 um diameter) at estimated 20 nm planar and 40 nm axial resolution. We will use novel microfluidic devices to effectively trap individual cells at multiple predefined position as to make them available for automated and parallelised super-resolution microscopy. This technique will allow efficient use of microscopy time and promises to shorten the overall experimental time. We will develop software to control our above microscopy instruments as modules for the open-source platform microManager and will make the source-code publically available.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING);2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);4.5 RESOURCES AND INFRASTRUCTURE (DETECTION);5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: GENERIC HEALTH RELEVANCE
Title: A hub for Engineering and exploiting the stem cell niche
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Our objectives and overall strategy is: (1) Through an understanding of normal tissue stem cell niches we will reconstruct the normal niche microenvironment to enable expansion and accurate differentiation of stem cells, and optimize small molecule and drug development strategies using adult stem cell, MSC, ES or iPS cell-based screening platforms. (2) To promote "healthy regeneration" we will identify the niche signals that control proliferation and differentiation of endogenous stem/progenitor cells within a diseased organ so as to facilitate the direct targeting of these cells using biological agents, drugs/small molecules or synthetic materials. This will utilise in vivo models or organ regeneration using zebrafish and then mouse models of organ injury and regeneration (3) To improve repair by transplanted cells we will understand and manipulate the abnormal niche created by the inflammation and damage of disease, as the milieu surrounding the transplanted cell has a profound effect upon both its longevity and function. We will utilise information from section (1) to facilitate this aim by providing the optimal defined niche around transplanted cells to make this successful.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.2 CELLULAR AND GENE THERAPIES
Health categories: GENERIC HEALTH RELEVANCE