ISOFLIM: Isotropic resolution fluorescence lifetime imaging of 3D neuron cultures

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Working at the very forefront of microscope development, this multidisciplinary research team aim to explore the four dimensions of space and time within live neurons. Using bioengineering, we have developed novel ways of training neurons to grow within specially created channels in biomaterials. The neurons make connections in these channels which enable us to investigate cell-to-cell communication in real-time as it would in the brain - in an entirely controllable way. Once we have grown these "wetware artificial neural networks" we can image their complex signalling behaviour using advanced microscopy. In this proposal, a new microscope concept will be developed which pushes the envelope of what can be seen at the cellular level. By creating a 3-dimensional lattice of optical foci in the sample and, in parallel, reading them out, we can create a 3D representation of the sample. Using ultra-sophisticated camera technology which was developed principally for 3D detection and ranging (LIDAR) in the automotive industry, called SPAD sensor arrays, we will measure the speed at which biological processes such as energy metabolism occur using a technique called fluorescence lifetime imaging microscopy (FLIM). FLIM is incredibly powerful for detecting changes in fluorescent molecules and can be used to measure protein-protein interactions or changes in protein conformation - essential processes for control of cellular behaviour.By adding fluorescent tags to proteins and illuminating them with a laser we can visualise them in a cell using SPAD sensor arrays. Energy transfer occurs when two of these tags with different colours come within a certain distance of each other, changing the amount of light that they emit. This Fluorescence Resonance Energy Transfer (FRET) can be measured to detect protein interactions. FLIM measures how the fluorescence lifetime changes during FRET and is not dependent on how much protein is present, making it a robust method for detecting protein interactions in live cells. The second difficulty in measuring FRET in moving cells, is that many imaging techniques are too slow and the amount of light from the laser can damage the cell. Our new microscopy method, ISO-FLIM (since it generates a isotropic resolution image), generates beams in a sheet of light that is shone onto the sample, which is recorded by a sensitive camera, making it fast and non-damaging to the cell. Our new method combines these techniques to create a new microscope to accurately and rapidly measure protein interactions in living neurons, allowing researchers to look at the 'real time' mechanics of protein function.