Development of Oblique Plane Microscopy for Biomedical Applications

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.

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.