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

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.

Neurodegenerative disorders are amongst the most impactful pathologies in modern society, and often feature loss of connectivity within neural circuits. Therefore, understanding the molecular basis of neuronal communication is crucial to elucidate their biological underpinnings. The convergence of bioengineering, stem cell technology and novel imaging reporters has enabled the creation of complex 3D in vitro platforms aimed at the study neuronal function (e.g. Ca2+ dynamics) in live human neurons and glia. These 3D cultures will soon represent the gold standard for in vitro modelling, and therefore it is necessary to ensure the field has the ideal tools to analyse them. Monitoring dynamic molecular processes in 3D cultures requires a quantitative based imaging system capable of acquiring large volumetric image data at high speed with minimal sample perturbation. Currently, such an imaging platform doesn't exist and compromises must be made between fast live imaging, super resolution and detailed quantitative imaging. To bridge this gap we aim to develop a novel functional imaging platform with fast 3D quantitative isotropic imaging to visualise calcium and metabolic dynamics within live neuronal 3D cultures. Building on previous work from the investigators, the system will (i) allow prolonged live imaging in 3D with low toxicity, (ii) enable high time- and spatial resolution, and (iii) provide for quantitative imaging data acquisition (e.g. fluorescence lifetime microscopy, FLIM). Utilising a combination of structured light sheet illumination and synchronous image scanning we will reconstruct selective planes with high spatial and temporal resolution using time-correlated single photon counting detection - giving the ultimate single photon sensitivity for FLIM (ISO-FLIM) in a light sheet geometry. The capability of the imaging system will be tested on a series of experiments with live stem cell derived bioengineered neural cultures with genetically encoded reporters

The proposed project aims to develop a massively parallel, high-speed isotropic selective plane Fluorescence lifetime imaging system and utilizing FRET based biosensors to monitor calcium dynamics, and metabolic activity in 3D bioengineered neuronal cell cultures in real-time. Once the proof-of-concept has been demonstrated this platform can be used to model and monitor other complex biological interactions and mechanisms. One of the main goals is to make it accessible to a wide variety of biological imaging problems from monitoring high-speed protein interaction dynamics to high-content screening applications using FRET readouts of cellular function or perturbation. The work proposed has the potential to have enormous impact to the clinical research community, industry and society.

Research Community

Increasingly sophisticated bio-engineered 3D structures which better mimic the complexity of neuronal circuitry in vivo coupled with the ability to optically interrogate and monitor those structures will undoubtedly have wide appeal to all experimental neuroscience and neuronal cell biology community. Although the biological exemplification for our proposal will be directly relevant for in vitro neuroscience, the protocols and imaging pipeline will immediately be applicable to any live imaging experiments, across fields such as cancer biology, molecular cell biology, microbiology and others (see diverse letters of support). It will also represent an invaluable tool for the bioengineering and tissue engineering community, as it will allow to evaluate dynamic molecular processes in live cells within 3D complex scaffolds.

Industry

From an economic perspective, the development of the ISO-FLIM imaging platform will be of great interest to the commercial imaging and life sciences market as well as the pharmaceutical industry. A number of instrumentation companies including Photon Force Ltd, Carl Zeiss Ltd and MSquared (see letters of support) are very interested in the system and would be the immediate beneficiaries (Ameer-Beg and Henderson have collaborative projects with Photon Force Ltd, through EPSRC Quantum hub (Quantic)and Carl Zeiss Ltd (BBSRC iCASE)). Any direct commercialization opportunities for software and hardware solutions that emerge from the research could be explored with these companies.

Pharmaceutical companies will be key longer-term beneficiaries as the activities provide a greater understanding of the consequences of molecular events at the system level leading to more refined drug discovery approaches (Ameer-Beg has key contacts in Pharma through previous collaborations and consultancy (UCB-Pharma and Novatis)). Many of the network members within the Integrative Biological Imaging Network (IBIN), who are strong supporters in this project, have long-term collaborations with the Pharmaceutical industry and will therefore be very well placed provide support to engage with these stakeholders as the network progresses to identify shared interests and collaboration opportunities.

Society

This project will generate a novel imaging platform aimed at dissecting complex molecular dynamics within living cells in 3D, and its applicability will be demonstrated in a series of proof-of-principle experiments based on advanced in vitro bioengineered neuronal cultures. As such, it will enable acquisition of functional data on these cells with speed and spatial resolution unmatched by current systems and therefore generate a wealth of new knowledge on the molecular processes involved in neuronal communication. In turn, this will immediately apply to experimental disease modelling with stem-cell derived in vitro culture, that are currently see their applicability limited by adequate characterization of their functional profile. The ISO-FLIM will be an enabling technology to apply new knowledge to neurodegenerative disease modelling, and directly lead to new potential therapies and patient benefit.