MICA: Bio-Continuum Microscopy: Seamless imaging from the micro- to the nano-scale View Homepage


Ontology type: schema:MonetaryGrant     


Grant Info

YEARS

2013-2017

FUNDING AMOUNT

1257654 GBP

ABSTRACT

Recent advances in biological imaging have focussed on improving resolution in both living and cryopreserved tissue. Despite exciting developments, the new techniques are still not ideal for many biological studies and remain far from simple to apply for non-specialists. To achieve a full understanding of the complex biological systems that underlie disease/infections and fundamental biology, it is imperative to understand all aspects of the mechanisms involved. Recent years have seen genomic/proteomic studies further our understanding, but we are still unable to visualise many key processes directly in an intact living cell. Thus, whilst recent developments are bringing us closer to this goal, a critical resolution gap between light and electron microscopy remains. Our electron-excited Super Resolution Microscopy (eSRM) technique will build on our Fluorescence Electron Microscopy (FEM) approach and seamlessly couple the technologies of light and electron microscopy to achieve a paradigm shift in biological imaging. This approach brings together the unique ability to image multiple coloured tagged-particles, such as antibodies, with the resolution of the electron microscope, whilst only requiring standard light microscopy preparations. It is therefore simple and easy to use across a broad range of biological questions. Preliminary work has established proof of concept that multicolour images can be acquired with a resolution of 3 nm on cell membranes. We are now uniquely placed to develop these methods and address vital medical/biological questions. Our ability to preserve fluorescent proteins (GFP) through sample preparation for electron imaging will revolutionise electron microscopy just as the same fluorophores did for light microscopy several decades ago. Our groundbreaking integration of SRM with both SEM and volume EM (3View) will allow us to exploit these developments quickly and efficiently to deliver accurate protein localisation in situ without complicated sample processing. Our strategy will have a major impact on the microscopy field; it will go beyond the information gained through recent advances and enable us to undertake molecular localisations and interactions under more relevant biological conditions. Current methods have a number of limitations that curtail data interpretation. For example, they rarely detect all the labelled proteins, require extensive imaging strategies and high resolution is usually achieved through mathematical algorithms rather than directly. Our techniques (eSRM, SRM coupled with CLEM) will enable studies at physiological concentration, visualise true molecular associations/distributions and permit study of fine-detailed sub-cellular and cell surface structures and changes in response to various stimuli. A key aspect of our approach is to enable easy sample handling and labelling so that non-specialist microscopists can use the methods. Our probes will be widely applicable to many microscopy methods, from in vivo multiphoton through to light emission from electron excitation. They are more photostable than existing probes and have very narrow emission bands, making multicolour imaging simpler. They have improved sensitivity and longer lifetimes which will be exploited when the very latest technologies are launched this year. In summary, by bridging the current gaps to produce a continuum light and electron microscopy approach and through combination of our latest advances, we aim to develop an integrated approach that far surpasses other techniques being developed. This will enable the step change required in microscopy to enable biologists to undertake true multicolour sub-light resolution imaging with very few constraints or specialist training, thus addressing a major limitation in biology and representing a ground-breaking advance in biological imaging. Technical Summary We will develop new imaging methods: 1 Integrate standard SRM with our novel electron-excited SRM making this technology simple to use. We will use our novel probes (see 3 below), which can be excited with both light and electron beams. Building on our expertise in standard SRM and CLEM, and our relationship with JEOL, we will improve the optical resolution of JEOL's novel ClairScope, a combined light/electron microscope. This will be achieved by adding a high performance PMT to the optical light path to enable ca 20 nm multicolour resolution. We will also build Bayesian Imaging onto the system for comparative imaging. 2 Develop integrated SRM-SEM for direct CLEM imaging. DELMIC will integrate PALM with the SECOM platform to study dual-label specimens in situ. Exploit our own recent advances in preserving fluorescent signals such that they can be used to give precise overlay of fluorescence and EM images. This will permit ca 2 nm resolution of samples. Finally we will integrate miniaturised fibre-optic laser microscopy into the automatic serial imaging workflow of the 3View platform to enable the analysis of complex networks in larger samples. 3 Further develop our novel probes. Optimise the probes for brightness and size distribution to be used in 1 and 2 (above) such that we will be able to image individual particles. We will also change the dopants so that the probes can also be used for live cell, MP imaging and then as fiducial markers for CLEM. These developments will address key biomedical questions, focussing on mammalian cell biology and molecular/cellular microbiology including -imaging of tissue, cellular and subcellular processes related to cancer. -visualizing activation of silent synapses related to learning and memory. -molecular/cellular microbiology questions related to bacterial and parasitic infections, the formation of bacterial communities and the mechanisms of bacterial chromosome segregation and plasmid partitioning. More... »

URL

http://gtr.rcuk.ac.uk/project/24746A16-2D2D-475D-8FB0-177A1970121E

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