ISOFLIM: Isotropic resolution fluorescence lifetime imaging of 3D neuron cultures
Lead Research Organisation:
King's College London
Department Name: Cancer Studies
Abstract
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.
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.
Technical Summary
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
Planned Impact
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.
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.
Publications
Balukova A
(2024)
Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models
in International Journal of Molecular Sciences
Chandler J
(2023)
In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.
in Proceedings of the National Academy of Sciences of the United States of America
Levitt JA
(2020)
Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM.
in Scientific reports
Yan Z
(2024)
FRET Sensor-Modified Synthetic Hydrogels for Real-Time Monitoring of Cell-Derived Matrix Metalloproteinase Activity using Fluorescence Lifetime Imaging
in Advanced Functional Materials
Description | We have developed a methodology for fluorescence lifetime imaging theta microscopy style light sheet methodology using two objectives mounted orthogonally with respect to the optical axis as outlined in the grant. The instrumentation is complex and we have had some delays in sourcing diffractive optical elements suitable for the task. In the last year, the PDRA associated with the grant has developed the optical instrumentation and integrated a cooled SPAD array detector. In addition a lattice illumination scheme has been developed based on a spatial light modulator grating pattern and interference between the zeroeth and first order diffraction patterns combined with a cylindrical lens. This intermediate imaging platform has the advantage of customisation with respect to the optical aberrations in the system. In addition, we have developed a novel 2-photon dual view imaging platform in both the up-right and inverted configurations ready to integrate the new SPAD arrays to be provided through the collaboration with Prof Robert Henderson at University of Edinburgh. Unfortunately, errors in prototype production of the SPAD arrays has significnatly delayed integration of the new SPAD arrays but will occur shortly. We have developed imaging protocols for fluorescence lifetime imaging of PersevalHR and Laconic biosensors for iPSC astrocyte cultures and these studies are on-going. |
Exploitation Route | Our newly developed dual view digitally scanned lightsheet platform is being further developed as a high-content imaging platform for a Wellcome LEAP consortium for time-lapse organoid imaging and will additionally be embedded within the Microscopy Innovation Center at KCL for user access. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | FLIMAGIN3D, EU MCSA-DN-2021, Marie Sklodowska-Curie Actions Doctoral Training Network |
Amount | £3,186,000 (GBP) |
Funding ID | 10107350 |
Organisation | Marie Sklodowska-Curie Actions |
Sector | Charity/Non Profit |
Country | Global |
Start | 01/2023 |
End | 01/2028 |
Description | Integrated multi-modal tissue state mapping of TNBC progression (WELLCOME LEAP) |
Amount | £4,200,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 08/2021 |
End | 09/2025 |
Description | Quantitative Multidimensional Imaging: A Centre of Excellence for Fluorescence Lifetime Imaging Microscopy |
Amount | £964,000 (GBP) |
Funding ID | MR/X012794/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2022 |
End | 03/2023 |
Title | Dual view digitally scanned light sheet system |
Description | A 2-photon excitation dual view light sheet system has been developed with a novel path selector and optimised optical design. The system enables imaging of 3D biological objects with a field of view of 300 microns cubed and with two colours sequentially. The system can be configured in both upright and inverted geometries dependent on the use case. |
Type Of Material | Technology assay or reagent |
Year Produced | 2023 |
Provided To Others? | No |
Impact | The system is a framework for future research and will be used to image a variety of biological models. The system is designed to integrate a SPAD array camera for fluorescence lifetime imaging which is currently being developed for deployment. |
Description | Collaboration to investigate cellular and molecular manipulation in an electro-kinetic trap |
Organisation | University Hospital Jena |
Country | Germany |
Sector | Hospitals |
PI Contribution | We have started collaboration with Prof Michael Boersch who is an expert in ATP synthase. We have contributed samples and technical expertise in FRET sensing of extracellular vesicles derived from cancer cells grown in the laboratory and with assays developed by the Ameer-Beg group. We are contributing expertise in optics and laser physics to improve an ABEL trap system for fluorescence lifetime imaging. We are investigating the use of SPAD arrays to further improve trapping feedback and real time analysis. |
Collaborator Contribution | Prof Boersch has contributed his time and analysis software in addition to access to his ABEL trap system to enable us to take measurements and investigate the possibility of improving the workflow and integrating our research methods into the platform. |
Impact | Research data has been gathered and some activities undertaken. Prof Ameer-Beg was invited to give a presentation at SPIE Photonics West 2024 to share details of this collaboration. |
Start Year | 2023 |
Description | Collaborative partnership to develop 3D projection diffractive optical element |
Organisation | Holoeye Photonics |
Country | Germany |
Sector | Private |
PI Contribution | We will develop new methods of measurement for three dimensional projected patterns generated by diffractive optical elements. We will help design new diffractive elements and compare these with the partner company We will test and measure diffractive optical patterns by projection with a spatial light modulator |
Collaborator Contribution | The partner will develop new Fourier optical devices for testing The partner will undertake modelling to optimise the Fourier patterns based on our testing and feedback The partner will manufacture binary diffractive elements for our testing and feedback |
Impact | Multidisciplinary: Optical Physics and applied optics |
Start Year | 2022 |
Description | Combining ErbB/HER Heterodimer Imaging Markers To Guide Trastuzumab Deruxtecan (T-DXd; DS-8201) Stratification |
Organisation | Daiichi Sankyo Company |
Country | Japan |
Sector | Private |
PI Contribution | This is a commercial partnership the details of which are partially confidential. However, in brief, we are analyzing the basis of drug resistance in a clinical compound. We are developing assays for FRET analsyis of protein-protein interactions which can be deployed in vitro cell culture and are working this assay up to use in 3D cell culture models using our advanced FLIM platforms. |
Collaborator Contribution | Diaichi have provided the drug and guidance with respect to desired outcomes. |
Impact | Too early. We have developed assays for protein interaction and are investigating mechanisms of drug resistance |
Start Year | 2023 |
Description | MSCA Doctoral Training Network: Flimagin3D |
Organisation | Trinity College Dublin |
Country | Ireland |
Sector | Academic/University |
PI Contribution | The MSCA-DTN includes two work packages that benefit from the technical developments generated during the ISOFLIM project. Light sheet platforms developed as part of the project are to be used by doctoral candidates at KCL and those on secondment at KCL from other European partners |
Collaborator Contribution | Partners will contribute their scientific expertise in 3D biological research models to explore optical imaging and functional analysis of cellular metabolism. |
Impact | No outputs ye, PhD candidates have been recruited and are working on developing 3D imaging platforms for metabolic research |
Start Year | 2023 |
Title | LUMINESCENCE IMAGING APPARATUS AND METHODS |
Description | Luminescence imaging apparatus, methods and computer program products are disclosed. A time-resolved luminescence imaging apparatus (100A) comprises: an optical assembly (2) operable to generate an array of beams; a scanner (4A) operable to scan the array of beams with respect to a sample (8), along a single scanning axis; and a detector assembly (10) having an array of detector elements, adjacent detector elements being spaced apart by an inter-element gap, each detector element being operable to detect emissions generated by the sample (8) in response to the array of beams. In this way, different locations on the sample (8) may be simultaneously scanned and imaged by the detector assembly (10) in order to image multiple parts of the sample (8) simultaneously. Also, by scanning along a single scanning axis, the complexity of the scanner (4A) is significantly reduced and the speed of scanning is increased compared to scanners which have to scan in two dimensions, such as a traditional raster scan mechanism. |
IP Reference | US2020132976 |
Protection | Patent application published |
Year Protection Granted | 2020 |
Licensed | No |
Impact | Nano Clinical Ltd has an option to licence subject to contract which will be optioned in the next 12 months. |
Title | LUMINESCENCE IMAGING APPARATUS AND METHODS |
Description | Luminescence imaging apparatus, methods and computer program products are disclosed. A time-resolved luminescence imaging apparatus (100A) comprises: an optical assembly (2) operable to generate an array of beams; a scanner (4A) operable to scan the array of beams with respect to a sample (8), along a single scanning axis; and a detector assembly (10) having an array of detector elements, adjacent detector elements being spaced apart by an inter-element gap, each detector element being operable to detect emissions generated by the sample (8) in response to the array of beams. In this way, different locations on the sample (8) may be simultaneously scanned and imaged by the detector assembly (10) in order to image multiple parts of the sample (8) simultaneously. Also, by scanning along a single scanning axis, the complexity of the scanner (4A) is significantly reduced and the speed of scanning is increased compared to scanners which have to scan in two dimensions, such as a traditional raster scan mechanism. |
IP Reference | WO2019008342 |
Protection | Patent application published |
Year Protection Granted | 2019 |
Licensed | No |
Impact | Nano Clinical Ltd has an option to Licence which will be optioned in the next 12 months. |
Company Name | Nanoclinical |
Description | Nanoclinical develops cancer treatments through the use of microscopy technology. |
Year Established | 2020 |
Impact | The company is just formed and does not yet have any impacts |
Website | https://www.nanoclinical.com/ |