A new generation of microscopes will soon resolve individual proteins inside living cells, revealing structures that have been invisible for centuries. Conventional light microscopes cannot see objects smaller than about 200 nanometres—roughly one five-hundredth the width of a human hair—because light waves blur together at that scale. This fundamental limit has hidden the molecular machinery that drives cell signalling, gene regulation, and immune responses. The project will build three different super-resolution microscopes at Imperial College London and the MRC Clinical Sciences Centre. One technique uses patterned light to double resolution; another uses a second laser beam to shrink the spot where fluorescence occurs, achieving detail below 50 nanometres; the third pinpoints individual fluorescent molecules one by one to reconstruct an image molecule by molecule. These instruments will let researchers watch how immune cells communicate, how nerve cells form synapses, how chromosomes organise inside developing germ cells, and how tumour cells interact with healthy tissue. The work is fundamental science aimed at understanding normal biology and disease mechanisms. If successful, it could eventually improve diagnosis of infertility, birth defects, and cancer by revealing the molecular events that drive them.
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Super-resolved microscopy (SRM) represents a revolution in optical imaging with profound implications for the life sciences. It allows researchers to visualise biological structure and function on almost the molecular scale in live cells and organisms. Typically biological processes are visualised by attaching fluorescent molecules to proteins of interest forming images from the light emitted by these fluorescent "labels". The optical resolution, which defines the smallest structures that can be visualised, has long been regarded as fundamentally limited to >200 nm by the diffraction of light waves. Recent breakthroughs in fluorescence microscopy, however, have exploited the ability to systematically switch the emission of fluorescent molecules on and off in order to break the "diffraction limit" and achieve resolutions down to 10's of nm - almost the scale of the typical biological proteins that are involved in signalling processes that determine the fate of cells and organisms and so are essential to disease processes. This project builds on the partnership between the MRC Clinical Sciences Centre (CSC) and Imperial College London to develop a range of SRM instruments to advance our understanding of epigenetics, regulation of genes and cells, immunology and metabolism by resolving fundamental biological mechanisms inside the nucleus, inside live cells and at the synapses of interacting cells. It also aims to establish super-resolved microscopy as a widely accessible tool for the research community at the CSC and Imperial. The first SRM technique would be structured illumination microscopy (SIM) that entails illuminating samples with patterns of light and observing the interference patterns between these patterns and the structure of the sample. This effectively doubles the resolution of a normal microscope. The second approach is an advance on laser scanning confocal microscopy, in which as sample is imaged by scanning a focused laser beam across a sample and collecting the fluorescence pixel by pixel through a pinhole that blocks out of focus or scattered light. The size of this focussed laser beam spot defines the resolution of this microscope. By using a second, collinear, specially shaped laser beam that is scanned synchronously and can switch off the fluorescence from molecules around the outside of the first beam focus, the fluorescence excitation is restricted to a smaller spot than the diffraction limit and thus resolutions below 50 nm can be realised. Because this technique can block scattered or out of focus light, it is particularly suitable for imaging in live organisms. The third SRM modality relies on having only a small fraction of the fluorescent labels being switched on at any time such that they can be treated like arrays of single molecules. Their individual positions can be determined by locating the centres of the "blobs" that represent each molecule in an image - which is possible to a precision of a few nm. By sequentially switching on different subsets of the fluorescent labels and determining their individual positions, one can build up a super-resolved "image" that is simply the map of the locations of all the fluorescent molecules. In this proposal, we will develop and apply these new imaging techniques to visualise biological structures and processes that are simply too small or inaccessible for conventional microscopes. Starting at the surface of cells, researchers will be able to observe how immune cells communicate and nerve cells interact with each other, and how tumour cells relate to normal cells. Looking inside cells, we will visualise signals that communicate the energy status of cells and how the genome is organised. It will be possible to resolve how chromosomes behave in developing germ cells, which is relevant to the most frequent causes of infertility and birth defects. The research will deliver new insights into both normal biological processes and disease states.
Amanda Fisher (Co-Investigator)Anthony Magee (Co-Investigator)Christopher Dunsby (Co-Investigator)David Carling (Co-Investigator)Enrique Martinez-Perez (Co-Investigator)Mark Neil (Co-Investigator)Matthias Merkenschlager (Principal Investigator)Paul Michael William French (Co-Investigator)Roy Taylor (Co-Investigator)Simona Parrinello (Co-Investigator)Vincenzo De Paola (Co-Investigator)
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