High content analysis of 3-D cell cultures with multidimensional fluorescence imaging
Lead Research Organisation:
Imperial College London
Department Name: Physics
Abstract
The response of cells to stimuli depends on cell signalling processes, which are combinations of molecular interactions. Disease is associated with deviations from normal signalling processes and so reading out molecular interactions in cells can help elucidate mechanisms of disease and also provide a means to evaluate potential therapeutic drugs. Many of the signalling molecules in cells are proteins and their interactions are widely studied using microscopy with proteins of interest being labelled with fluorescent molecules ("fluorophores"). For drug discovery, fluorescence imaging of arrays of cells is automated so that the effect of many compounds on cell signalling processes can be "assayed". Fluorophores are "excited" by radiation at a wavelength at which they absorb and the resulting characteristic emission (fluorescence) is recorded using an imaging detector. By labelling different kinds of protein with different fluorophores and comparing the images at their respective emission wavelengths, it is possible to learn about protein interactions by observing when they appear in the same place at the same time. Unfortunately this "co-localisation" is usually limited by diffraction of light to a resolution of a few 100 nm - much larger than the size of typical signalling proteins (~1-10 nm). It is possible, however, to confirm interactions using Förster Resonant Energy transfer (FRET), which entails labelling the proteins with different fluorophores and observing when energy is transferred between them. This energy transfer can only occur if they are within ~10 nm of each other. The most quantitative way to read out interactions using FRET is through the reduction in the fluorescence lifetime of the "donor" fluorophore as it loses energy due to the energy transfer. Fluorescence lifetime measurements can be made in every pixel and this fluorescence lifetime imaging (FLIM) enables protein interactions to be mapped in space and time. Fluorescence lifetime can also be used to distinguish different molecular species or different states of naturally fluorescent molecules that are involved in regulating the consumption of energy in the cell, which can also be altered by disease.
For cell biology research and for drug discovery, fluorescence-based studies typically involve imaging thin - and therefore transparent - layers of cells. Unfortunately this is not a physiologically normal environment for cells and they often behave differently compared to when they are in their normal 3-D tissue context. However, it is highly challenging to image cells in native biological tissue and even more so to realise this in a high throughput mode for drug testing. Instead, there is increasing interest in assaying synthetic 3-D cultures comprising many cells that interact with each other, presenting behaviour reminiscent of that in native tissue with greater optically accessibility - although unfortunately they can scatter and absorb light more strongly than thin layers of cells. Such 3-D cell cultures can be arrayed for rapid imaging, however, and we propose to develop an automated platform to provide 3-D images of such cell cultures, utilising FLIM to read out molecular interactions. For this we will optimise the 3-D cell culture and labelling methodologies for fluorescence imaging and will develop and evaluate automated microscopes for FLIM-based assays. For larger samples we will utilise multiphoton excitation, which entails illuminating the fluorophores at twice the wavelength usually required for excitation, such that they need two photons arriving simultaneously. This two photon absorption is intensity-dependent and so can be arranged to occur only in the focal plane of a scanning laser beam such that the emitted photons all originate from a specific depth in the sample. Scanning the focal plane then enables 3-D imaging. The longer wavelength light is less phototoxic and is scattered less by the sample, thus enabling deeper imaging.
For cell biology research and for drug discovery, fluorescence-based studies typically involve imaging thin - and therefore transparent - layers of cells. Unfortunately this is not a physiologically normal environment for cells and they often behave differently compared to when they are in their normal 3-D tissue context. However, it is highly challenging to image cells in native biological tissue and even more so to realise this in a high throughput mode for drug testing. Instead, there is increasing interest in assaying synthetic 3-D cultures comprising many cells that interact with each other, presenting behaviour reminiscent of that in native tissue with greater optically accessibility - although unfortunately they can scatter and absorb light more strongly than thin layers of cells. Such 3-D cell cultures can be arrayed for rapid imaging, however, and we propose to develop an automated platform to provide 3-D images of such cell cultures, utilising FLIM to read out molecular interactions. For this we will optimise the 3-D cell culture and labelling methodologies for fluorescence imaging and will develop and evaluate automated microscopes for FLIM-based assays. For larger samples we will utilise multiphoton excitation, which entails illuminating the fluorophores at twice the wavelength usually required for excitation, such that they need two photons arriving simultaneously. This two photon absorption is intensity-dependent and so can be arranged to occur only in the focal plane of a scanning laser beam such that the emitted photons all originate from a specific depth in the sample. Scanning the focal plane then enables 3-D imaging. The longer wavelength light is less phototoxic and is scattered less by the sample, thus enabling deeper imaging.
Technical Summary
3-D cell cultures are being explored for a range of assays and research studies, including cancer progression and stem cell differentiation, since their signalling processes are expected to be much closer to the in vivo context than for conventional monolayer cell cultures. We aim to develop an automated multidimensional fluorescence imaging platform for assays of signalling processes in 3-D cell cultures, particularly tumour spheroids, including FLIM for quantitating protein interactions and changes in cellular metabolism. Unfortunately, their increased scattering, aberration and attenuation of optical signals make them more challenging to image than 2-D cell cultures. With GSK, we propose to explore and develop high content tools and methodologies for image-based assays of cell signalling and morphological changes in 3-D cell cultures. Building on our experience developing automated FLIM multiwell plate readers, we propose to take advantage of the inherently ratiometric nature of fluorescence lifetime measurements that enables quantitative molecular readouts in spite of scattering and attenuation of excitation and fluorescence emission - as already exploited for intravital imaging. In particular, we would use FLIM to quantify FRET readouts of protein interactions and genetically expressed biosensors as well as autofluorescence of NADH and collagen. We would investigate trade-offs between 3-D cell culture methods, sample preparation, imaging speed and functional readouts for exemplar assays of protein multimerisation, of genetically expressed FRET biosensors and autofluorescent metabolites and of protein-histone association. This project would include spinning disc confocal and multibeam multiphoton microscopy to image spheroids of different sizes and at different speeds, in order to give GSK (and other pharma) insights into optical approaches for assaying 3-D cell cultures.
Planned Impact
The proposed technology platform and methodology should benefit researchers wanting to understand cell signalling processes and disease mechanisms and clinicians and pharma wanting to develop therapies since the 3-D cell cultures are believed to resemble "normal" behaviour in vivo more closely than the thin layers of cells cultured on transparent substrates that are mainly used for microscopy in cell biology research and assays for drug discovery. From discussions with our pharma colleagues (GSK and AZ) and at live cell assay meetings (e.g. Informa Life Sciences Drug Discovery Innovations, Berlin 2013; SMi's 6th annual Cell Based Assays, London 2013), there is increasing interest in drug testing with 3-D cell cultures including tumour spheroids, which would specifically benefit cancer studies, and stem cell cultures that can be used for a broad spectrum of applications including regenerative medicine.
Improving the physiological relevance of assays could impact the efficiency of the drug discovery pipeline, perhaps on a 5 year timescale, and ultimately reduce its cost - with further economic and societal benefits. It could also reduce the need for animal testing. Unfortunately, it is not usually considered practical to screen with 3-D cell cultures, partly because their higher absorption and scattering and less reproducible growth compared to monolayers of cells make high content analysis more challenging. While today there are a few vendors selling systems for growing 3-D cell cultures, they are usually imaged with conventional plate readers that provide wide-field images or sectioned images near the surface of, e.g. tumour spheroids and we are not aware of any automated assays of cell signalling in 3-D cell cultures. It is therefore of immediate interest to GSK and other pharma to better understand the potential for using 3-D cell cultures in drug discovery and the most direct impact would come from providing this information to GSK and subsequently to other pharma through presentations at conferences, including pharma industry-orientated meetings where the applicants are often invited to speak. Increasing the efficiency of the drug discovery pipeline would lead to economic benefits (over 5-15 years) for pharma and related industrial sectors. Following the impact on pharma, the beneficiaries of more efficient drug discovery would include patients benefitting from improved therapies and healthcare providers benefitting from cheaper, more effective drugs and fewer therapeutic interventions. This societal impact would be on a timescale of >10 years.
The specific biology applications in the project could impact research across a wide range of diseases including cancer, diseases such as diabetes associated with metabolism and obesity and autoimmune diseases, with concomitant benefits to patients, healthcare providers and industry in the longer term. The ability to read out molecular interactions, metabolic changes and the formation of complex tissue structures in 3-D cell culture could be important for other fields of biomedical research and industry including tissue engineering, regenerative medicine and monitoring of other therapeutic approaches such as radiation therapy. For the technologies and methodologies developed in this project to be accessible by pharma, other industry and academe, they need to be commercially available. As this project highlights important potential applications of 3-D cell cultures, it would present opportunities for instrumentation and software companies to address these potential markets, thereby generating economic impact over a ~3-10 year timescale.
This project would provide world-class training for the RAs in imaging technology and software, cell-based assays and the biology of 3-D cell cultures, all of which are in demand and could help establish careers in academic or commercial research and development as well as more general careers in management, consulting and technical sales.
Improving the physiological relevance of assays could impact the efficiency of the drug discovery pipeline, perhaps on a 5 year timescale, and ultimately reduce its cost - with further economic and societal benefits. It could also reduce the need for animal testing. Unfortunately, it is not usually considered practical to screen with 3-D cell cultures, partly because their higher absorption and scattering and less reproducible growth compared to monolayers of cells make high content analysis more challenging. While today there are a few vendors selling systems for growing 3-D cell cultures, they are usually imaged with conventional plate readers that provide wide-field images or sectioned images near the surface of, e.g. tumour spheroids and we are not aware of any automated assays of cell signalling in 3-D cell cultures. It is therefore of immediate interest to GSK and other pharma to better understand the potential for using 3-D cell cultures in drug discovery and the most direct impact would come from providing this information to GSK and subsequently to other pharma through presentations at conferences, including pharma industry-orientated meetings where the applicants are often invited to speak. Increasing the efficiency of the drug discovery pipeline would lead to economic benefits (over 5-15 years) for pharma and related industrial sectors. Following the impact on pharma, the beneficiaries of more efficient drug discovery would include patients benefitting from improved therapies and healthcare providers benefitting from cheaper, more effective drugs and fewer therapeutic interventions. This societal impact would be on a timescale of >10 years.
The specific biology applications in the project could impact research across a wide range of diseases including cancer, diseases such as diabetes associated with metabolism and obesity and autoimmune diseases, with concomitant benefits to patients, healthcare providers and industry in the longer term. The ability to read out molecular interactions, metabolic changes and the formation of complex tissue structures in 3-D cell culture could be important for other fields of biomedical research and industry including tissue engineering, regenerative medicine and monitoring of other therapeutic approaches such as radiation therapy. For the technologies and methodologies developed in this project to be accessible by pharma, other industry and academe, they need to be commercially available. As this project highlights important potential applications of 3-D cell cultures, it would present opportunities for instrumentation and software companies to address these potential markets, thereby generating economic impact over a ~3-10 year timescale.
This project would provide world-class training for the RAs in imaging technology and software, cell-based assays and the biology of 3-D cell cultures, all of which are in demand and could help establish careers in academic or commercial research and development as well as more general careers in management, consulting and technical sales.
Publications
Alexandrov Y
(2018)
Quantitative time domain analysis of lifetime-based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions.
in Journal of biophotonics
Chennell G
(2016)
Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM.
in Sensors (Basel, Switzerland)
Fang Z
(2020)
The Influence of Peptide Context on Signaling and Trafficking of Glucagon-like Peptide-1 Receptor Biased Agonists.
in ACS pharmacology & translational science
French P
(2017)
Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
in Journal of Visualized Experiments
Garcia E
(2020)
FLIM, FRET and high content analysis
Guglielmi L
(2021)
Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways.
in Nature communications
Görlitz F
(2017)
Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.
in Journal of visualized experiments : JoVE
Jones B
(2021)
Genetic and biased agonist-mediated reductions in ß-arrestin recruitment prolong cAMP signaling at glucagon family receptors.
in The Journal of biological chemistry
Maioli V
(2016)
Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.
in Scientific reports
Title | MOESM1 of In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime |
Description | Additional file 1: Figure S1. Schematic representation of the optical set-up of multidimensional spectrofluorometer as described in Manning et al. [41]. |
Type Of Art | Film/Video/Animation |
Year Produced | 2017 |
URL | https://springernature.figshare.com/articles/presentation/MOESM1_of_In_vivo_label-free_mapping_of_th... |
Title | MOESM1 of In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime |
Description | Additional file 1: Figure S1. Schematic representation of the optical set-up of multidimensional spectrofluorometer as described in Manning et al. [41]. |
Type Of Art | Film/Video/Animation |
Year Produced | 2017 |
URL | https://springernature.figshare.com/articles/presentation/MOESM1_of_In_vivo_label-free_mapping_of_th... |
Title | MOESM2 of In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime |
Description | Additional file 2: Figure S2. Distribution of fluorescence lifetimes in untreated Triticum aestivum plants of different age groups calculated from multispectral lifetime point-probe measurements in the spectral channel CH4 (excitation at 440 nm, detection wavelengths 620-710 nm). Data points from different age groups are represented by different colours. Plant samples are named in the format p(n)l(m), where n is the plant number and m is the leaf number. The weighted mean fluorescence lifetime (tm) calculated for each sample is plotted here. |
Type Of Art | Film/Video/Animation |
Year Produced | 2017 |
URL | https://springernature.figshare.com/articles/presentation/MOESM2_of_In_vivo_label-free_mapping_of_th... |
Title | MOESM2 of In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime |
Description | Additional file 2: Figure S2. Distribution of fluorescence lifetimes in untreated Triticum aestivum plants of different age groups calculated from multispectral lifetime point-probe measurements in the spectral channel CH4 (excitation at 440 nm, detection wavelengths 620-710 nm). Data points from different age groups are represented by different colours. Plant samples are named in the format p(n)l(m), where n is the plant number and m is the leaf number. The weighted mean fluorescence lifetime (tm) calculated for each sample is plotted here. |
Type Of Art | Film/Video/Animation |
Year Produced | 2017 |
URL | https://springernature.figshare.com/articles/presentation/MOESM2_of_In_vivo_label-free_mapping_of_th... |
Description | We have established that we can apply FLIM to cells in 3-D culture and can obtain quantitative readouts of FRET biosensors, including the T2-AMPKAR FRET biosensor. This work has been presented at SLAS2018 in San Diego and will be presented at 2018 SLAS Advanced 3D Human Models and High-Content Analysis Conference, Leiden. We have developed four different approaches to producing 3-D cell cultures and managed to grow spheroids of NIH3T3 cells for the first time. We have worked to establish the limits of performance using our optically sectioned 3-D multiwell plate FLIM FRET instrument. We find we can apply quantitative FLIM to a depth of ~40 microns in tumour spheroids and then can be limited by out of focus light. We have established that we can image deeper - potentially to a depth >80 microns using multiphoton excitation for FLIM. We then demonstrated a multiwell plate FLIM microscope utilising multibeam multiphoton microscopy (based on a LaVision Biotec TriMScope) and applied this to imaging spheroids expressing a fluorescent protein (FP)-based FRET construct. We have also applied a novel light sheet microscope implantation (oblique plane microscopy) to tumour spheroids and shown that we can obtain FRET readouts with spectral ratiometric imaging. Using appropriate controls, we have established that our FLIM readouts are quantitative (providing a uniform 3D response where no heterogeneity is expected) and that we can image heterogeneity in the response of FRET biosensors expressed in tumour spheroids where it is present. Quantitative FLIM readouts, e.g. of FRET are achievable at greater depths where the individual cells are not resolved. We have also extended FLIM FRET multiwell plate assays by implementing it with open source software (automated FLIM data acquisition controlled using MicroManager and FLIM data analysis using our open source MATLAB plugin: FLIMfit). We have also published a JoVE paper providing a detailed description of how to use our software and have provided a list of components to enable other groups to duplicate out work. In response to a challenge from pharma, we have also successfully demonstrated the application of automated multiwell plate FLIM to assay endogenous proteins labelled with FP - rather than using plasmids to over-express FRET constructs. This is important because over-expressed proteins may not faithfully represent the underlying biology that is to be studied using FLIM FRET. We have also addressed the issue of possible artefacts in FLIM FRET data due to the low rotational diffusion time of FP that means their emission is better modelled by a static random isotropic distribution of fluorophore dipoles - rather than the usual assumption of dynamic averaging of a random isotropic distribution of fluorophore dipoles during the fluorescence decay. We developed a software tool that can take the fluorescence lifetime data from conventional (incorrect) analysis of FP-based FLIM FRET data and estimate what would be the "true lifetime parameters assuming a static random isotropic distribution of fluorophore dipoles. This is openly shared (URL given below) The expertise we gained in building automated multiwell plate imaging instrumentation for high content analysis (HCA) has been extended beyond FLIM/FRET and we have developed a widely applicable MicroManager-based software tool that we are currently also applying to automated super-resolved HCA using STORM. |
Exploitation Route | We believe we are developing a new assay platform that will be of use to biologists and to drug discovery since the ability to undertake FLIM/FRET assays of 3D cell cultures provides a more physiologically realistic context in which to assay cell signalling processes. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
URL | https://github.com/yalexand/dk2_to_sk2_calculator |
Description | We worked with GSK to explore the utility of a FRET-based assay in tumour spheroids. We were able to culture the spheroids expressing a FRET biosensor and undertake an automated multiwell plate FLIM FRET assay. This contributed to GSK knowledge of the potential of this specific biosensor and the whole project added to their knowledge of FLIM/FRET assays in 3D cell culture. |
First Year Of Impact | 2016 |
Sector | Pharmaceuticals and Medical Biotechnology |
Description | Accelerating our ability to understand and target complexity and heterogeneity in cancer through automated imaging of 3D cancer models including patient derived organoids |
Amount | £4,515,484 (GBP) |
Funding ID | C10441/A29368 and C10441/A30808 |
Organisation | Cancer Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2020 |
End | 02/2025 |
Description | BBSRC Impact Acceleration Account: Open, modular, accessible, super-resolved microscopy |
Amount | £47,212 (GBP) |
Funding ID | BB/S506667/1. |
Organisation | Imperial College London |
Sector | Academic/University |
Country | United Kingdom |
Start | 05/2018 |
End | 11/2019 |
Description | High content FLIM, FRET, and localisation imaging to characterise inter-cellular heterogeneity in PI3K signalling to glycolysis in breast cancer and improve treatment strategies |
Amount | £485,352 (GBP) |
Funding ID | A28450 |
Organisation | Cancer Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 08/2019 |
End | 07/2022 |
Description | Imperial Confidence in Concept (ICiC) Scheme |
Amount | £75,000 (GBP) |
Organisation | Imperial College London |
Sector | Academic/University |
Country | United Kingdom |
Start | 07/2019 |
End | 12/2020 |
Description | Research England GCRF fund: openScope - phase 1 |
Amount | £174,089 (GBP) |
Organisation | Imperial College London |
Sector | Academic/University |
Country | United Kingdom |
Start | 11/2018 |
End | 09/2019 |
Description | Research England devolved Global Challenges Research funding |
Amount | £132,933 (GBP) |
Organisation | United Kingdom Research and Innovation |
Department | Global Challenges Research Fund |
Sector | Public |
Country | United Kingdom |
Start | 07/2019 |
End | 07/2020 |
Description | Astrazeneca |
Organisation | AstraZeneca |
Country | United Kingdom |
Sector | Private |
PI Contribution | We undertook a collaborative project with AZ at the Francis Crick Institute: Determination of heterogeneity of response to different inhibitors in EGFR mutant Non-small lung adenocarcinoma cells using HCA-FLIM, |
Collaborator Contribution | We used the knowhow and some instrumentation developed during the BBSRC funded project for the automated FRET microscopy we undertook for this project. In-kind contributions from AZ were mainly advice and co-design of project. |
Impact | This collaboration was multidisciplinary. The main outcomes were insights into how to further develop assays for study heterogeneity in response to drugs |
Start Year | 2017 |
Description | Cairn Research Ltd |
Organisation | CAIRN Research Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | We have developed a range of microscopy techniques aimed to widen access to advanced microscopy, including for scientists from LMIC and other lower resourced settings. We have published the openFrame concept, CAD files and component lists under open -source licences. Cairn have implemented these ideas into their product development and will be able to provide the benefit of low-cost but advanced microscopy techniques to the community. These include research-grade fluorescence microscopes based on our modular openFrame concept, optical projection tomography and an optical autofocus system. |
Collaborator Contribution | Cairn have advised on components and practical implementations of various aspects of our instruments and have provided us with prototypes at significant discounts that have been customised to our requirements. We have advised them on key research issues for microscopy and designed open source optical instrumentation for which they are now making components available on a commercial basis to the wider user community who wish to access the technology but cannot or do not wish to fabricate it themselves. This instrumentation is intended to work with open source software, including our open source software. Cairn will now provide our openFrame microscope that can be used to implement almost any other microscope modality at relatively low cost and with potential to upgrade. openFrame is our original concept and the design has been refined with input from Cairn. We are currently co-designing a new microscope incubator compatible with openFrame microscopes and HCA ancillary instrumentation. |
Impact | The instrumentation development is essentially optical physics but the applications are multidisciplinary - mainly biomedical |
Start Year | 2015 |
Description | GSK |
Organisation | GlaxoSmithKline (GSK) |
Country | Global |
Sector | Private |
PI Contribution | We have worked with GSK since 2006 when we collaborated on a DTI Technology project to develop automated multiwell plate FLIM FRET assay technology. Currently we are working with GSK as part of the BBSRC IPA project to determine whether we can use our FLIM FRET platform to assay bromodomain histone binding using a genetically expressed FRET biosensor and compare inhibitor compound effects between 2-D and 3-D assays. We have developed this assay in 2D cell culture with both transient transfection and in stable cell lines and obtained first results. We are working to extend this to 3D cell culture. |
Collaborator Contribution | GSK have provided us with plasmids for the bromodomain-histone binding FRET sensor and advice to help us generate the stable cell line. They have helped us review results and plan future work. They have also made a contribution of £57876 towards the project. |
Impact | Since 2006, this collaboration led to the current BBSRC IPA project being funded. BBSRC provide expertise in drug discovery and we provide expertise in biophotonics instrumentation, particularly multiwell plate FLIM FRET. |
Start Year | 2006 |
Description | Kentech Instruments Ltd |
Organisation | Kentech Instruments Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | We have advised Kentech on improvements to be made to their gated optical image intensifiers and related components and have tested prototypes. We have demonstrated that their gated intensifiers can be integrated into a range of optical instruments, particularly for fluorescence lifetime imaging (FLIM) for biomedical applications, and this has led to increased sales for them. We collaborated with them on an EPSRC Healthcare Partnership project that built on earlier collaborations with Kentech and also on two MRC projects to develop FLIM instrumentation. |
Collaborator Contribution | Kentech Instruments Ltd advised us (with no charge) on the design of FLIM systems using gated optical image intensifiers and supplied us with a custom device for handheld FLIM that we designed in collaboration. They have sold us customised gated intensifier systems and refined the specifications of their products at our request, which has impact all our subsequent research involving wide-field time-gated FLIM. |
Impact | Joint publications Feedback to Kentech's product development |
Title | FLIMfit |
Description | New open source fluorescence lifetime fitting tool with unprecedented functionality and speed ?FLIMfit? developed and released through Open Microscopy Environment http://www.openmicroscopy.org/site/products/partner/flimfit This software includes global fitting tools to fit the complex fluorescence decay profiles often encountered when analysing FRET data derived from fluorescence proteins in biosensors. |
Type Of Technology | Software |
Year Produced | 2017 |
Open Source License? | Yes |
Impact | This FLIMfit software has been downloaded and is in use in many locations including the UK Europe, USA and Australia. It has been used by us for FLIM/FRET high content analysis and for optical projection tomography. It has been continuously refined and updated until the present (2017). |
URL | http://www.openmicroscopy.org/site/products/partner/flimfit |
Title | Multibeam multiphoton multiwell plate microscope |
Description | This is an automated multiphoton microscope designed to image 2D and 3D cancer models and other biological samples arrayed in multiwell plates for high content analysis. It is built on our openFrame platform and integrated open-source and commercial products. |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2024 |
Impact | We have not published this yet but are currently applying it to study the action of anticancer drugs. |
Title | openFLIM HCA: Automated multiwell plate FLIM microscope for optically sectioned assays in 3-D cell culture including time-lapse FLIM/FRET studies in live cells |
Description | This automated multiwell plate FLIM microscope is controlled by open source (MIcroManager) software and the component lists are shared on our website to enable other research groups to replicate our instrumentation. FLIM data analysis, including global fitting, is provided by our open source software tool: FLIMfit, written in MATLAB. Exciation is provided by a super-continuum source or a frequency-doubled mode-locked Ti:Sapphire laser. FLIM detection is realised though time-gated imaging using a gated image intensifier. This instrument typically acquires a field of view every 10 seconds, including the time for sample movement and autofocus. |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2016 |
Impact | This instrument has been applied to a number of applications including protein interactions of the kinetochore, metabolic changes in differentiating embryonic stem cells, bromodomain-histone binding and a genetically expressed FRET biosensor for EPAC,a Rap-1 guanine exchange factor that binds specifically to cAMP. |
URL | http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/openflim-hca/ |
Title | openHCA-FLIM µManager Plugin |
Description | This is the (MicroManager Plugin) control software for our openFLIM HCA instrument that provides automated multiwell plate FLIM, with the option to implement optical sectioning. |
Type Of Technology | Software |
Year Produced | 2016 |
Impact | This open source software is freely available and should enable other research groups to build their own FLIM HCA instrumentation. |
URL | http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/openflim-hca/ |
Title | sk2 FRET calculator |
Description | We developed a way to analyse fluorescence lifetime FRET data recorded from FRET experiments using fluorescent proteins that takes into account their slow rotational dephasing (which contradicts the standard analysis used for FLIM FRET data). We developed an approach that can fit fluorescence lifetime data to the appropriate model or can correct fluorescence lifetime parameters obtained from the conventional but incorrect analysis approach. The latter capability is available as an open source MATLAB tool. |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2018 |
Impact | This attracted interst in the community and let us being invited to present and demonstrate our software at a FRET workshoip in Warsaw. |
URL | https://github.com/yalexand/dk2_to_sk2_calculator |
Description | MDFI user-orientated workshop 2017 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | This one day user-orientated workshop entitled "Multidimensional fluorescence imaging technology: super-resolution, HCA and preclinical imaging" showcased the biophotonics instrumentation and applications of the technologies developed in the Photonics Group at Imperial College London. It attracted 80 participants including 11 from industry and 40 for other research organisation. The talks and posters were well received and this event led to further collaborations, including with industry. |
Year(s) Of Engagement Activity | 2017 |
Description | SMi conference on 3D Cell Culture |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | This is a commercially organised conference attended mainly by people working in the pharmaceutical industry. |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.smi-online.co.uk/pharmaceuticals/uk/conference/3D-Cell-Culture |
Description | SRM workshop at Hammersmith Hospital 01/03/17 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | We organised a workshop to show case the new super-resolved microscopy and FLIM/FRET microscopy capabilities that we have developed during this project. We presented the technical advances and the capabilities that our life scientists could use for their research. We also arranged for our life scientist colleagues to explain how they have been using the technology and how they would like it to further develop. |
Year(s) Of Engagement Activity | 2017 |
Description | School visit (Horsted Keynes) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | I presented the impact of biophotonics and imaging technology on the world around us and on medicine and drug discovery |
Year(s) Of Engagement Activity | 2017 |