A Superresolution microscope for biological research in the multi-user Microscopy Facility [FILM]: ALERT 13
Lead Research Organisation:
Imperial College London
Department Name: National Heart and Lung Institute
Abstract
Unlike most analytical methods, light microscopy allows the investigation of biological processes in a non-destructive way, in their natural context. Unfortunately, it is limited by the diffraction limit of light, which only allows visualisation of structures ~10x the size of individual molecules. However, when a cell responds to environmental signals (e.g. growth factor response, immune response, differentiation), essential decisions depend on the interaction of single or few molecules. Electron microscopy is able to look at individual molecules, but requires extensive fixation and staining protocols incompatible with living cells. Recently, new imaging techniques have been developed that circumvent the diffraction limit of light: They all take advantage of the fact the diffraction limits the size of structures that can be resolved, but much smaller structures can be imaged as long as they emit enough light. One powerful method is localisation microscopy, which achieves this by sequentially imaging single (or few) molecules - sufficiently spaced from each other - which are then switched off. The localisation of these molecules can then be determined with high precision, and from many (10,000-30,000) frames, a high-resolution image can be constructed. This method offers theoretically unlimited (and practically ~10-fold) resolution, but is limited to 2D or small 3D volumes and is slow, so only suited for fixed or slow-moving live samples. We therefore want to combine it with another method which allows full 3D super-resolution at high speed: Illuminating the sample with a grid pattern will result in an interference pattern from the low frequency (large scale) of the illumination pattern and the high frequency (small scale) of the sub-resolution structures in the sample. From the known structure / frequency of the grid pattern, the unknown structure of sub-resolution structures can be mathematically calculated. This method is fast and works in full 3D, but is limited in its resolution to 2x the normal resolution.
In the current proposal, we propose to install a new, fully automated instrument combining both methods, thus allowing high-resolution, full-3D, fast imaging of fixed and live samples with multiple fluorescent labels. Success of these methods depends critically on sample preparation, of which we have years of experience in the light microscopy facility of Imperial College [FILM], based on a simple, manual super-resolution microscope. The results also critically depend on the correct processing and interpretation of the data, as well as handling of huge amounts of image data. We therefore propose to extend our existing image data workflow around the OMERO open-source image database and use it as the central hub for super-resolution data, from which the raw data are sent off for processing and the results written back and linked to the original data, allowing efficient, central processing, easy comparison of multiple settings and algorithms and full documentation. Eventually, published data including raw data will also be made available through OMERO.
In the current proposal, we propose to install a new, fully automated instrument combining both methods, thus allowing high-resolution, full-3D, fast imaging of fixed and live samples with multiple fluorescent labels. Success of these methods depends critically on sample preparation, of which we have years of experience in the light microscopy facility of Imperial College [FILM], based on a simple, manual super-resolution microscope. The results also critically depend on the correct processing and interpretation of the data, as well as handling of huge amounts of image data. We therefore propose to extend our existing image data workflow around the OMERO open-source image database and use it as the central hub for super-resolution data, from which the raw data are sent off for processing and the results written back and linked to the original data, allowing efficient, central processing, easy comparison of multiple settings and algorithms and full documentation. Eventually, published data including raw data will also be made available through OMERO.
Technical Summary
Microscope specifications:
Dual-mode super-resolution microscope (SRM) with localisation-based SRM (PALM, STORM) and structured illumination (SIM) capability. Additional specifications:
Total Internal Reflection (TIRF) illumination and objective lens
Three high-power imaging lasers (plus low-power switching laser)
Fast piezo focus
Infrared Autofocus
Temperature / CO2 incubation
Motorised stage
High-sensitivity, parallel cameras with optical wavelength separation
Parallel two-colour imaging
Accessories
room upgrades (Air conditioning)
Cell culture equipment
Support:
Dedicated staff during installation and training
Data storage and processing upgrade
Dual-mode super-resolution microscope (SRM) with localisation-based SRM (PALM, STORM) and structured illumination (SIM) capability. Additional specifications:
Total Internal Reflection (TIRF) illumination and objective lens
Three high-power imaging lasers (plus low-power switching laser)
Fast piezo focus
Infrared Autofocus
Temperature / CO2 incubation
Motorised stage
High-sensitivity, parallel cameras with optical wavelength separation
Parallel two-colour imaging
Accessories
room upgrades (Air conditioning)
Cell culture equipment
Support:
Dedicated staff during installation and training
Data storage and processing upgrade
Planned Impact
The proposed super-resolution equipment would have a direct impact on a wide range of research projects in Imperial College. In addition, the equipment would be of particular relevance for the researchers in the Centre for Integrated Systems Biology and Bioinformatics, a co-founder of the light microscopy facility (in its former form of the BBSRC-funded CISBIC) and long-term user and supporter of the facility. Because of the power of novel microscopy techniques to understand biological processes in a comprehensive way down to the single-molecule level, the expertise of the light microscopy facility [FILM] in leading edge technologies is also regularly a decisive contributor to attract highly qualified young researchers to Imperial College.
Imperial College has also an outstanding relationship with commercial partners in the UK and worldwide. Imperial Consultants is the UK's leading academic consultancy provider, and FILM is the partner facility of Imperial Consultants covering business requests in the area of light microscopy. Super-resolution has only recently started to move from front-line technological development to mainstream application to solve biological problems, but the complexity of the technology requires a thorough understanding of the whole experimental workflow, from the choice of the best technology through to fluorescent labelling, sample preparation, image acquisition parameters, data processing parameters, data analysis and data visualisation. FILM has developed solid expertise in all these aspects, and a leading-edge super-resolution instrument would offer an attractive business opportunity for the UK biotechnology industry.
One commercial entity to profit directly from the new equipment would be the pharmaceutical company Emergent Biosolutions, which incorporates Microscience, an Imperial College spin-off co-founded by Professor David Holden. Microscience was founded to develop novel vaccines based on the research tools developed in David Holden's lab, which studies host-pathogen interactions important for human and animal health (Streptococcus, Staphylococcus, Salmonella). Understanding and visualising host-pathogens interactions on the molecular level could open new insights which could be explored for the development of novel vaccines to animal diseases.
In a similar way, all other research projects included in this proposal study molecular interactions essential for understanding the response of cells to their environment, with regard to host-pathogen interactions, tumour biology, stem cell developments. All these areas are directly relevant to animal and human health, be it immunobiology, tissue regeneration, anti-malarial strategies or anti-cancer drugs.
Beyond the scientific impact, we are also proposing to develop a robust solution to handle the large amount of data involved in super-resolution microscopy. We plan to use OMERO as the primary data hub. Imperial College is a partner institution for the integration of OMERO in local academic institutions. We plan to use OMERO as a central hub to link raw data with processing tools and localisation results in an efficient and reliable way, so that raw data, processing parameters and results are kept together and well documented for future reference. The resulting workflow would then be made available to the scientific community as an open-source tool. Such a tool has the potential to (1) improve the quality of data by improving transparency and workflow for the end-user biologist, (2) stimulate good scientific practice by comparing algorithms, (3) stimulate data exchange and collaboration by better documentation of published data and (4) save considerable amounts of data storage costs for researchers, institutes and funding bodies and researchers by minimising data duplication.
Imperial College has also an outstanding relationship with commercial partners in the UK and worldwide. Imperial Consultants is the UK's leading academic consultancy provider, and FILM is the partner facility of Imperial Consultants covering business requests in the area of light microscopy. Super-resolution has only recently started to move from front-line technological development to mainstream application to solve biological problems, but the complexity of the technology requires a thorough understanding of the whole experimental workflow, from the choice of the best technology through to fluorescent labelling, sample preparation, image acquisition parameters, data processing parameters, data analysis and data visualisation. FILM has developed solid expertise in all these aspects, and a leading-edge super-resolution instrument would offer an attractive business opportunity for the UK biotechnology industry.
One commercial entity to profit directly from the new equipment would be the pharmaceutical company Emergent Biosolutions, which incorporates Microscience, an Imperial College spin-off co-founded by Professor David Holden. Microscience was founded to develop novel vaccines based on the research tools developed in David Holden's lab, which studies host-pathogen interactions important for human and animal health (Streptococcus, Staphylococcus, Salmonella). Understanding and visualising host-pathogens interactions on the molecular level could open new insights which could be explored for the development of novel vaccines to animal diseases.
In a similar way, all other research projects included in this proposal study molecular interactions essential for understanding the response of cells to their environment, with regard to host-pathogen interactions, tumour biology, stem cell developments. All these areas are directly relevant to animal and human health, be it immunobiology, tissue regeneration, anti-malarial strategies or anti-cancer drugs.
Beyond the scientific impact, we are also proposing to develop a robust solution to handle the large amount of data involved in super-resolution microscopy. We plan to use OMERO as the primary data hub. Imperial College is a partner institution for the integration of OMERO in local academic institutions. We plan to use OMERO as a central hub to link raw data with processing tools and localisation results in an efficient and reliable way, so that raw data, processing parameters and results are kept together and well documented for future reference. The resulting workflow would then be made available to the scientific community as an open-source tool. Such a tool has the potential to (1) improve the quality of data by improving transparency and workflow for the end-user biologist, (2) stimulate good scientific practice by comparing algorithms, (3) stimulate data exchange and collaboration by better documentation of published data and (4) save considerable amounts of data storage costs for researchers, institutes and funding bodies and researchers by minimising data duplication.
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| Description | The grant was for a piece of equipment (a superresolution microscope) in a multiuser microscopy facility (FILM). It has been extensively used by several users and has contributed to their research which is reported elsewhere. |
| Exploitation Route | The equipment has yielded important results for users and will continue to do so going forwards |
| Sectors | Agriculture, Food and Drink,Chemicals,Education,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | MRC Confidence in Concept Scheme and Wellcome Trust Institutional Strategic Support Fund |
| Amount | £57,920 (GBP) |
| Funding ID | PS3085_CHBBT and PS3110_CHBBC |
| Organisation | Medical Research Council (MRC) |
| Department | MRC Confidence in Concept Scheme |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 10/2017 |
| End | 10/2018 |
| Description | Multi-User Equipment grant |
| Amount | £703,000 (GBP) |
| Funding ID | WT104931MA |
| Organisation | Wellcome Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 10/2014 |
| End | 12/2019 |
| Description | molecular imaging collaboration with Neil MA, Dunsby C, French PM |
| Organisation | Imperial College London |
| Department | Department of Physics |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | providing biological samples and context for imaging using state-of-the-art and novel microscopy techniques |
| Collaborator Contribution | they provided access to state-of-the-art and novel microscopic imaging modalities and collaboration in applying them |
| Impact | Publications PMID: 19343675; 19550853; 17728161; 16617080; 20540668; multidisciplinary - cell biology and photonics |