Development of Oblique Plane Microscopy for Biomedical Applications
Lead Research Organisation:
Imperial College London
Department Name: Physics
Abstract
Conventional optical microscopy uses visible light to provide high resolution (>~200 nm) images for a huge range of applications from industrial inspection of electronic devices to biomedical imaging of cells and tissue. Fluorescence microscopy is an extension of optical microscopy that has become a standard tool for biologists who label their specimens with fluorescent molecules to report the locations of specific proteins, or to study cellular processes, e.g. by imaging proteins interacting.Many microscopy applications demand optically sectioned imaging, which provides an image of only a single thin (~1 micron) slice through the sample. Optical sectioning improves image contrast by reducing the 'blur' from out-of-focus planes that is evident in conventional microscopy and provides the ability to produce 3D images from stacks of 2D optically sectioned images. Normally, optically sectioned imaging is implemented using expensive laser scanning confocal microscope systems (typically >150k), which can be considered as a gold standard . While these microscopes provide high resolution 3D images, they typically require 10's of seconds to acquire a 3D fluorescence intensity image. Also, when imaging at higher speeds, the illumination used in confocal microscopes can cause light induced damage (photodamage) to biological samples.Several alternative optical sectioning microscopy techniques have been developed that generally address some of the disadvantages of confocal microscopy, but which inevitably present a compromise elsewhere. One recently developed alternative is selective plane illumination microscopy (SPIM), which provides rapid optically sectioned imaging and with very low exposure of the sample to illumination light, and which is particularly advantageous when imaging live biological specimens. However, a major disadvantage of SPIM is that it cannot be implemented on the standard fluorescence microscopes that are used widely in biomedical research.This project will develop a new optically sectioning microscope technology invented by the applicant called Oblique Plane Microscopy (OPM, patent filed July 2008). OPM is conceptually similar to SPIM but it can be implemented on standard fluorescence microscopes and applied to image samples prepared on standard microscope slides or the standard cell culture dishes or multiwell plates that are used by the vast majority of biologists. As for SPIM, the image acquisition rate of OPM is only limited by the speed of the CCD camera used (e.g. 1000 frames per second) and OPM subjects the specimen to a minimal light exposure. This project will apply OPM to two different biological applications. The first will image isolated beating heart muscle cells to measure and quantify the propagation of calcium waves and to image small rapid changes in calcium concentration known as 'calcium sparks'. OPM will provide high speed optically sectioned fluorescence detection to image spark events and will also be used for time-lapse 3D imaging of calcium wave propagation within individual heart muscle cells. The second biological application will be to image small (~50 micron) fluorescently labelled zebra fish embryos. The results obtained on these biological samples will demonstrate the utility of OPM and provide preliminary data for future interdisciplinary research projects.A key advantage of OPM is the potential for high-speed high-throughput automated 3D imaging, e.g. in multi-well plates used in biological and drug-discovery assays. This project aims to demonstrate this potential, which is important for commercial exploitation. A further route to commercialisation of OPM could be as a 'bolt-on unit' to a conventional fluorescence microscope that could provide optically sectioned imaging at significantly lower cost than a laser scanning confocal microscope.
Planned Impact
This proposal is an essential step towards the development and commercialisation of a new, widely enabling, imaging technology for research and drug discovery that will benefit UK science in a competitive and strategically significant area and will also result in the interdisciplinary training of a postdoctoral researcher with a physical science background in biomedical applications of imaging. Oblique plane microscopy (OPM) will provide new capabilities for high speed optically sectioned imaging and time-lapse 3D fluorescence imaging and will therefore benefit academic and commercial researchers working in the fields of biology and medicine who use optically sectioning fluorescence microscopy techniques as a tool in their research. This is likely to be achieved through a microscope bolt-on unit that can be added to existing microscope systems, therefore lowering the cost of ownership compared to confocal microscopes and, more importantly, providing reduced photobleaching and phototoxicity for 3D imaging. Because OPM can be implemented on a standard fluorescence microscope and be applied to standard preparations of biological cells or organisms, it is directly applicable in current biological laboratories, unlike the technique of selective plane illumination microscopy (SPIM). As a result, OPM will find applications in multiwell plate assays, which are currently used in high content drug screening by the pharmaceutical industry, and will lead to new 3D fluorescence screening methods. This would continue the current trend in drug discovery towards High Content Analysis and would also be applicable to higher-throughput biology experiments. In the longer term, there are potential applications of OPM in the field of cytometry, which is typically performed in either a flow or an imaging configuration, i.e. flow cytometry or quantitative automated imaging cytometry. Cytometry is used in the pharmaceutical industry, basic biomedical research and for clinical screening applications, e.g. for blood born disease. OPM, when combined with high speed computer based image analysis, will be able to provide quantitative multiparameter 3D imaging of cells flowing in microfluidic channels or, equivalently, similar imaging information in a 3D automated imaging cytometer configuration. Both would allow a greater range of morphological and biochemical parameters to be studied and would increase the amount and quality of information obtained. These applications will be best realized if OPM can be commercialised as a new microscope or microscope attachment. As a result, this research will benefit companies manufacturing microscope equipment. Any exploitation of the research will benefit the UK economy via the patent application filed through Imperial Innovations plc. The preferred route for commercialization of this technology would be a licensing agreement with an established microscope manufacturer. One of the international market leaders has already expressed some interest and is carrying out an internal assessment of the technology but the market research and negotiating position would be much more effective once the biological utility of the technique has been demonstrated, which is a key goal of this proposal. There are several UK SME's that may have an interest in commercialising OPM as an add-on to existing microscope frames and the proposed preliminary biological data could support a subsequent collaborative development programme, perhaps funded by EPSRC and/or the TSB.
People |
ORCID iD |
Christopher Dunsby (Principal Investigator) |
Publications
Dunsby C.
High Speed Volumetric Imaging by Oblique Plane Microscopy
in 1st Light Sheet Fluorescence Microscopy International Conference, Barcelona, 25th Sept 2014
Dunsby C.
High speed 2D and 3D fluorescence by oblique plane microscopy
in Crick Symposium: New Frontiers in Optical Microscopy
Kumar S
(2011)
High-speed 2D and 3D fluorescence microscopy of cardiac myocytes.
in Optics express
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
Sikkel M
(2016)
High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes
in Journal of Biophotonics
Description | Oblique Plane Microscopy (OPM) is a novel fluorescence microscopy technique that can be used to image fluorescent biological specimens. The key advantages of OPM are that it exposes the specimen to only very low levels of illumination, thus reducing the unwanted effects of light induced destruction of the fluorophores being studied (photobleaching) and light induced damage of a live specimen (phototoxicity and photodamage). In addition, it can acquire an image of a single slice in the specimen without the need for moving parts, enabling very high speed 2D and 3D imaging. This project successfully implemented the technique of OPM as a module that can be attached to a conventional inverted fluorescence microscope frame for the first time. This has the significant advantage that biologists using the microscope can first locate their specimen using conventional microscopy techniques, such as bright-field microscopy, phase contrast microscopy or epi-fluorescence microscopy, and then switch to using OPM of the same field of view. Together with a new high speed and highly sensitive camera, these developments enabled OPM to be used successfully to study high speed events in biological specimens. The first specimens that were studied were isolated heart cells in collaboration with Dr Ken MacLeod and Dr Alex Lyon at the National Heart and Lung Institute. The cells were first loaded with a fluorescent calcium indicator that reports changes in the local calcium concentration through changes in its brightness. Using OPM, we imaged the calcium dynamics in these cells of electrically stimulated contractions, spontaneous calcium waves and small localised calcium events called sparks. Usually, biologists use 1D line-scanning microscopy to study these rapid spark events and the new OPM system now permits them to be imaged in 2D at up to 926 frames per second, allowing both the localisation and dynamics of these small rapid events to be investigated. The study of calcium dynamics in heart cells, and especially spark dynamics, is important to further biologists' understanding of the processes occurring in heart cells that are linked to heart failure. The new OPM system also enables high speed volumetric microscopy at up to 30 volumes per second, which allows calcium dynamics to be studied in 3D for the first time. We used the OPM system to image spontaneous calcium waves propagating through single cardiac myocytes in 3D, allowing us to precisely pinpoint the origin of the wave. In a second collaboration with Professor Maggie Dallman's group in the Division of Cell and Molecular Biology at Imperial, the OPM system was used to perform 3D imaging of zebrafish embryos. Through the use of a motorized scanning stage and image-stitching it was possible to image a whole embryo in 3D in only 2 minutes. Using this approach, we performed a preliminary study to quantify the 3D tumour volume in a zebrafish model. |
Exploitation Route | OPM has a number of potential applications in industry, for example: • In the pharmaceutical industry for high-speed 3D fluorescence imaging in High Content Screening of cells and/or embryos in multiwell plate formats or high throughput 3D imaging of non-adherent cells in flow cytometry type configurations. • For the characterisation of fluid flow dynamics in 3D in microfluidic devices. The results from this project have led directly to follow-on research funding awarded by the BBSRC (BB/I023801/1), which aims to further increase the image acquisition speed, number of laser excitation wavelengths and detection channels available to biologists using the system for the study of heart disease in isolated cardiac myocytes. There is also a growing list of collaborators within Imperial from Biology, the National Heart and Lung Institute and Bioengineering who want to use this technology. The results from this project have also been crucial in the successful licensing of this technology to industry. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology,Other |
URL | http://www3.imperial.ac.uk/photonics/research/biomedical-imaging/obliqueplanemicroscopy |
Description | The results from this project have led directly to follow-on research funding awarded by the BBSRC (BB/I023801/1), which aims to further increase the image acquisition speed, number of laser excitation wavelengths and detection channels available to biologists using the system for the study of heart disease in isolated cardiac myocytes. There is also a growing list of collaborators within Imperial from Biology, the National Heart and Lung Institute and Bioengineering who want to use this technology. The IP associated with this project has been licenced by Leica Microsystems.through Imperial Innovations. |
Sector | Healthcare,Pharmaceuticals and Medical Biotechnology,Other |
Impact Types | Economic |
Description | BBSRC Grouped |
Amount | £148,319 (GBP) |
Funding ID | BB/I023801/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2011 |
End | 07/2012 |
Description | EPSRC, Imperial College London Impact Acceleration Account, Pathways to Impact |
Amount | £76,896 (GBP) |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 12/2015 |
End | 03/2017 |
Description | Multidisciplinary Project Award - co-funded by EPSRC |
Amount | £495,000 (GBP) |
Funding ID | C53737/A24342 |
Organisation | Cancer Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2017 |
End | 09/2020 |
Description | Novel optical approaches to understanding the microscopic origins of calcium waves and the mechanisms underlying their arrythmogenic properties |
Amount | £283,307 (GBP) |
Funding ID | NH/16/1/32447 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2017 |
End | 03/2020 |
Description | Collaboration with NHLI on Oblique Plane Microscopy |
Organisation | Imperial College London |
Department | National Heart & Lung Institute (NHLI) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We developed a novel light-sheet based microscopy techinque called Oblique Plane Microscopy (OPM) |
Collaborator Contribution | OPM was applied to quantifying calcium dynamics in cardiomyocytes with collaborators at the NHLI. |
Impact | This collaboration resulted in two funded research projects: EP/H03238X/1 and BB/I023801/1 |
Start Year | 2010 |
Title | Licensing of IP |
Description | IP associated with the invention of Oblique Plane Microscopy was licenced to a commercial company. |
IP Reference | |
Protection | Patent application published |
Year Protection Granted | |
Licensed | Yes |
Impact | First live-cell 3-D time-lapse imaging of calcium wave origins in cardiomyocytes. |
Description | Light sheet microscopy for high content 3-D imaging of 3-D tissue cultures in a 96-well plate format |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Oral presentation at SLAS, Washington, 2017 |
Year(s) Of Engagement Activity | 2017 |
URL | http://www.slas2017.org/ |
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 | The Oblique Plane Microscope Plate-Reader |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | STEM for Britain - a poster competition held at the Houses of Parliament for early career researchers. |
Year(s) Of Engagement Activity | 2017 |
URL | http://www.setforbritain.org.uk/index.asp |