Advanced live imaging for the Eastern ARC with dual inverted light-sheets and AI-led analysis
Lead Research Organisation:
University of Essex
Department Name: Biological Sciences
Abstract
Microscopy has turned into an incredibly exciting area of research over the last two decades, with two recent Nobel Prizes and many new technological developments. Scientists can now observe cells moving around, digesting, interacting, multiplying and assembling at high resolution and over long periods. But cells are easily damaged if intense light is focussed on them. So when observing cells with a microscope, it is important to minimise such photodamage by lowering the light intensity. However, lower levels of light result in poorer images in the microscope and less useful information, so there is a clear dilemma between high light (good image, but cells damaged) and low light (cells healthy but image worse). Lightsheet microscopy, a relatively new technique, can solve this issue by providing good images at low light intensity: Delicate cells, tissues and whole organisms are scanned with a 'sheet' or plane of light and can be observed over days, producing breathtaking movies. Together with collaborators across the Eastern Arc, which comprise the universities of Essex, Kent and East Anglia, we want to use a high-end light-sheet microscope in combination with cutting-edge software to address new and exciting biological questions. This will allow many research groups in the South East and East of England to use light-sheet microscopy for their investigations. By establishing a nation-wide light-sheet user group to share new ways to use light-sheet microscopes. By making this technology more accessible to users, imaging with light sheets is poised to give us new insights into biological systems in ways we may not have thought possible.
Technical Summary
Within the Eastern Academic Research Consortium (Eastern Arc), comprising the universities of Essex, Kent and East Anglia, we want to use a unique light-sheet microscope, the Marianas LightSheet (MLS) by 3i, together with best-in-class image processing and analysis software Aivia. The MLS is a powerful imaging approach that enables high-speed, high-resolution imaging of live samples at low-to-no phototoxicity, with optical sectioning and large fields of view. It will be applied to functional and developmental imaging in a wide range of model systems. Powerful bioimaging technologies need to be available at a local level. This is particularly true for affordable technologies that have the potential to be widespread and are slowly finding their way into more general, non-specialist research settings, such as light-sheet fluorescence microscopy. In the South East and East, coverage of LSFM is sparse. This is a significant regional gap of LSFM expertise and instrument availability. An instrument at the University of Essex, where the main applicant is a highly research- and training-active member of the light-sheet community, would be an important first step towards filling this gap. We have the expertise and facilities to conduct the original research projects detailed in this application, establishing innovative ways to image important biological model systems that lie within the BBSRC's strategic priorities.
| Description | The light-sheet microscope obtained through this funding is enabling us to image important living samples from diverse fields. We are currently focussing on biomedically and environmentally relevant biofilm development as well as plant physiology. This light-sheet microscope allows detailed - and importantly very gentle and non-invasive - imaging of these samples in three dimensions and over long periods of time. The (very large) datasets we obtain are a powerful basis to model growth and behaviour of these samples in different conditions. |
| Exploitation Route | We continue to demonstrate the importance of minimally- to non-invasive live imaging as a fundamentally important 'best practice'. The acquired data (and quantification approaches) are crucial for modelling biofilm growth (by mathematicians and computer scientists) and a deeper understanding of plant signalling. |
| Sectors | Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology Other |
| Description | Raising awareness of phototoxicity |
| Geographic Reach | Multiple continents/international |
| Policy Influence Type | Influenced training of practitioners or researchers |
| URL | https://quarep.org/working-groups/wg-13-phototoxicity/ |
| Description | High resolution determination of multi species biofilm development on tracheostomy tubing |
| Amount | £105,984 (GBP) |
| Funding ID | BB/X512199/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 08/2022 |
| End | 09/2026 |
| Description | ROS signaling in plants: Are we missing a fundamental pathway? |
| Amount | £198,380 (GBP) |
| Funding ID | BB/Y513362/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2024 |
| End | 01/2026 |
| Title | Sensitive method for assessing phototoxicity in fluorescence microscopy |
| Description | Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells. This study addresses the need for relatively simple and sensitive methods to establish a dose-response curve for phototoxicity in mammalian cell line models. We conclude with a working model for phototoxicity and recommendations for its assessment. |
| Type Of Material | Model of mechanisms or symptoms - in vitro |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | A crucial consideration for imaging live cells is how to assess phototoxicity in a given sample. Different approaches have been used and greatly vary in their sensitivity. Readouts range from viability (live/dead cells) and cellular morphology on the rather blunt end of the assessment criteria spectrum to the dynamics of a biological process and gene expression on the sensitive readout end. In this study, we use a sensitive and straight-forward method to establish a dose-response curve for phototoxicity in mammalian cell line models. Specifically, we assess the effect of blue light, as commonly used for GFP and YFP excitation, on a motile mammalian cell line. Phototoxic effects are measured using a sensitive dynamic process, showing that motility can be used to reliably assess subtle positive and negative effects of illumination. For highly sensitive detection of the effect of blue light, we analysed the expression of genes involved in oxidative stress. |
| URL | https://www.frontiersin.org/articles/10.3389/fcell.2021.738786/full |
| Description | EasternARC Imaging Platform Alliance |
| Organisation | University of East Anglia |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Establishment of a framework to share large, strategic and platform equipment between the Eastern Arc universities (University of Essex, University of Kent and University of East Anglia). Each of the universities has significant microscopes and other imaging facilities (such as the recently-awarded lightsheet microscope at Essex), and making these available will both enable world class research to take place, but also facilitate collaborations and closer links between colleagues at three universities. by which colleagues can access and use equipment at each of the universities. The Alliance sees this being put into practice. ' The MoU relates to large, strategic equipment and facilities, and makes clear that the Alliance should be as unbureaucratic as possible. 'This will be crucial,' said Ward. 'If the process is difficult and drawn out, people won't engage. It should be as easy to access equipment at a partner institution as it is at your own.' A working group is currently working to finalise these details, and it is hoped that the Alliance will launch in 2023. |
| Collaborator Contribution | Memorandum of understanding (MoU) for the development of an Imaging Platform Alliance signed by the PVCs of the EasternARC universities (July 2022). |
| Impact | Regular meetings between the EasternARC universities to establish a framework. |
| Start Year | 2022 |
| Description | EasternARC Imaging Platform Alliance |
| Organisation | University of Kent |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Establishment of a framework to share large, strategic and platform equipment between the Eastern Arc universities (University of Essex, University of Kent and University of East Anglia). Each of the universities has significant microscopes and other imaging facilities (such as the recently-awarded lightsheet microscope at Essex), and making these available will both enable world class research to take place, but also facilitate collaborations and closer links between colleagues at three universities. by which colleagues can access and use equipment at each of the universities. The Alliance sees this being put into practice. ' The MoU relates to large, strategic equipment and facilities, and makes clear that the Alliance should be as unbureaucratic as possible. 'This will be crucial,' said Ward. 'If the process is difficult and drawn out, people won't engage. It should be as easy to access equipment at a partner institution as it is at your own.' A working group is currently working to finalise these details, and it is hoped that the Alliance will launch in 2023. |
| Collaborator Contribution | Memorandum of understanding (MoU) for the development of an Imaging Platform Alliance signed by the PVCs of the EasternARC universities (July 2022). |
| Impact | Regular meetings between the EasternARC universities to establish a framework. |
| Start Year | 2022 |
| Description | Understanding the mechanisms that inhibit and promote biofilm expansion. |
| Organisation | University of Adelaide |
| Country | Australia |
| Sector | Academic/University |
| PI Contribution | In collaboration with Prof Campbell Gourlay (University of Kent, UK), we are providing microscopy imaging datasets of growing biofilms. Many of these were acquired with the light-sheet microscope obtained with this grant. With my group at the University of Essex, we further provide image processing expertise and algorithms that are required to extract quantitative data from the live imaging datasets. |
| Collaborator Contribution | Binder, Benjamin J. is Professor at the University of Adelaide and has received funding for this work (grant reference: DP230100406) as principal investigator. Our group comprises leading researchers from Adelaide, Southampton, Kent and Essex (ASKE) Universities. We are developing mathematical and experimental models of yeast colony growth. Our interdisciplinary approach deepens understanding of fundamental growth mechanisms and their implications for biofilm-forming yeasts of medical and technological importance. |
| Impact | We are working on data analysis and modelling, and so far have had many internal meetings, but not yet any published outcomes. This collaboration is highly interdisciplinary, comprising bespoke molecular biology and sample preparation approaches as well as advanced image acquisition and image processing to provide the quantified basis for mathematical models of biofilm growth. Our interdisciplinary approach deepens understanding of fundamental growth mechanisms and their implications for biofilm-forming yeasts of medical and technological importance. |
| Start Year | 2023 |
| Description | Understanding the mechanisms that inhibit and promote biofilm expansion. |
| Organisation | University of Kent |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | In collaboration with Prof Campbell Gourlay (University of Kent, UK), we are providing microscopy imaging datasets of growing biofilms. Many of these were acquired with the light-sheet microscope obtained with this grant. With my group at the University of Essex, we further provide image processing expertise and algorithms that are required to extract quantitative data from the live imaging datasets. |
| Collaborator Contribution | Binder, Benjamin J. is Professor at the University of Adelaide and has received funding for this work (grant reference: DP230100406) as principal investigator. Our group comprises leading researchers from Adelaide, Southampton, Kent and Essex (ASKE) Universities. We are developing mathematical and experimental models of yeast colony growth. Our interdisciplinary approach deepens understanding of fundamental growth mechanisms and their implications for biofilm-forming yeasts of medical and technological importance. |
| Impact | We are working on data analysis and modelling, and so far have had many internal meetings, but not yet any published outcomes. This collaboration is highly interdisciplinary, comprising bespoke molecular biology and sample preparation approaches as well as advanced image acquisition and image processing to provide the quantified basis for mathematical models of biofilm growth. Our interdisciplinary approach deepens understanding of fundamental growth mechanisms and their implications for biofilm-forming yeasts of medical and technological importance. |
| Start Year | 2023 |
| Description | Chair for Phototoxicity working group (WG13), Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy. Since May 2022. |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Key aims of WG13 are: - To bring together the international microscopy community to raise awareness about the issue of phototoxicity in live fluorescence microscopy. This includes imaging scientists working in core facilities, researchers and trainees (graduate students, postdoctoral fellows), scientific publishers, manuscript/grant reviewers, and manufacturers of optical instruments and software. - The WG members will generate content and give phototoxicity workshops and short talks at different scientific meetings in their regional area and in their scientific field (e.g. biophysics, neuroscience). WG13 will also periodically produce educational material (blog articles, videos, reviews) with up-to-date references (peer-reviewed publications, white papers and industry notes) regarding phototoxicity. - To develop and disseminate general recommendations and best practices on how to measure, mitigate and report phototoxicity. For example, how to produce dose-response curves, how to avoid illumination overhead, and what biological readouts to use. - To establish a consensus on phototoxicity-related terminology. For these points, we intend to work with the Microscopy Publication Standards group (WG11). - To work with the Illumination Power group (WG1) to encourage researchers to measure incident light power for all live cell experiments. - To collaborate with industry partners for the promotion of hardware settings and techniques that verifiably enable live imaging with low phototoxicity. - To obtain funding that enables quantitative studies, educational activities and community building centered on phototoxicity. |
| Year(s) Of Engagement Activity | 2022,2023,2024 |
| URL | https://quarep.org/working-groups/ |
| Description | Various international workshops (ELMI 2024, EuBI 2023, SPAOM 2021, MMC (2021 & 2020)) on phototoxicity in fluorescence microscopy. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Workshops (mainly online) for wide audiences of life science researchers (students, group leaders, industry members) on how to identify, minimise and report phototoxicity. Each workshop had around 50-100 attendants. |
| Year(s) Of Engagement Activity | 2020,2021,2022,2023,2024 |