A super-resolution microscopy platform for imaging cells at multiple spatial scales

Fluorescence imaging has allowed researchers to explore the localisation, dynamics and plasticity of structures within cells. Although revolutionary at the time, the resolution of classical fluorescence imaging microscopes is constrained by the diffraction limit of light. This means that the precise location and organisation of molecules in a cell cannot be accurately visualised. Instead, only a blurred description of their whereabouts was possible, without any possibility of uncovering how single molecules were arranged in space. The advent super-resolution microscopy has changed all this - it is now possible to resolve the location of individual molecules using fluorescence imaging and begin to explore a whole new world of molecular arrangements that takes place at the nanoscale. In fact, these new approaches have been instrumental in uncovering novel sub-cellular structures within cells, which will help better understand their function. To delve into this new world of the very small, it is important to have the right equipment that allows single-molecule imaging. In particular, it needs to be able to do so for molecules tagged with different fluorescent compounds to uncover multi-molecular structures, with high precision in 3D and over large fields of view to encompass as much information as possible. Finally, it is important to also have access to imaging tools that give low resolution maps of the molecules of interest. In this way, we aim to provide large-scale descriptions of the distribution of molecules in cells and neurons and subsequently home into some of these domains to characterise them at very high spatial resolution. Here, we propose to use this approach to answer basic cell biology questions in different systems, including in intact tissue slices, to establish the properties of synapses formed by neurons in the brain and understand the local structure of the cytoskeleton in cells grown in vitro.

Single-molecule localisation microscopy (SMLM) comprise a group of techniques that have allowed researchers to image the location of molecules with a spatial resolution that overcomes the diffraction limit of light. Here we propose to acquire a the Abbelight fluorescence microscope that provides the highest 3D resolution of any commercially available system, across multiple fluorophores, without chromatic aberration and over large fields of view, to uncover structures that were previously invisible to non-super-resolved imaging platforms. When coupled to a confocal imaging system that can rapidly acquire low resolution images over large areas, it opens up the possibility of characterising molecular structures within cells across multiple spatial scales. Existing commercial microscopes that carry out SMLM techniques typically only allow imaging of a single fluorophore, with lower resolution in the z-axis and over a small field of view. In addition, commercial SMLM platforms do not include confocal imaging capabilities. The Abbelight SAFe 360 microscope will not only strengthen King's position as a global leader in fluorescence imaging approaches, but will also provide the scientific community at King's and the broader London region with the possibility of exploring new research avenues that deal with the nanoscale world of subcellular domains.