Development of a multi-channel Oblique Plane Microscopy system for cardiac cell physiology measurements

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. to image the local calcium concentration in cells. 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. One recently developed alternative to confocal microscopy is selective plane illumination microscopy (SPIM), which provides rapid optically sectioned imaging, very low exposure of the sample to illumination light, and which is therefore 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 without significant modification of the method used to mount the sample. This project will develop a new optically sectioning microscope technology invented by Dr Chris Dunsby 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, tissue culture dishes or multiwell plates. As with SPIM, the image acquisition rate of OPM is only limited by the speed of the CCD camera used and OPM subjects the specimen to a minimal light exposure. To date, OPM has been successfully demonstrated for high-speed optically sectioned imaging of calcium waves in single live isolated cardiac myocytes at 500 frames per second and these images offer an exceptional view of calcium dynamics. However, these successful initial experiments immediately highlight a clear gap in the capability of the existing system: that it is not possible to image multiple cellular parameters simultaneously and that only one excitation wavelength is available. Therefore, the primary goal of this project is to extend the existing OPM system to enable multiple fluorescent probes to be studied simultaneously through the addition of laser excitation sources at new wavelengths and the addition of multiple detection channels that can image at different wavelengths using state of the art and extremely high speed camera technology. Significantly, the improved OPM system will be able to directly study multiple cellular parameters, e.g. the intracellular sites of calcium release will be correlated with the organization and structure of the cell membrane during cell contraction - in both 2 and 3 spatial dimensions - for the first time on a rapid timescale. While the focus of this proposal is to apply the improved OPM system to imaging cardiac cell dynamics, it will also be applicable to a number of other important imaging challenges in biology, including high-speed 3D imaging in cardiac tissue slices and for monitoring the diffusion and clustering of proteins on the cell membrane in 2D on the millisecond timescale.

This project will improve and extend a novel optically sectioning fluorescence microscopy technique called oblique plane microscopy (OPM). The proposed modifications are essential to allow OPM to be used for cutting-edge biological research, and will be tailored specifically to the demands of studying dynamic events in cardiac myocytes. Two state-of-the-art high speed scientific CMOS cameras will be added to the OPM system to allow high speed imaging in two spectral channels simultaneously, which is essential if two fluorescent probes are to be studied and correlated, or if quantitative ratiometric imaging of e.g. [Ca2+] is to be achieved. Multichannel imaging and ratiometric imaging will greatly enhance the potential of OPM to produce novel biological results. The improved OPM system will offer simultaneous two-channel time-lapse 2D imaging with up to 528x512 pixels at 400 fps, or at up to 144x128 pixels at 1600 fps, which will offer an unprecedented insight into cardiac cell dynamics on timescales previously restricted to 1D line-scanning confocal microscopy. When combined with the ability of OPM to rapidly scan the observation plane through the sample without the need to move either the sample or the primary microscope objective, then the modified OPM system will also be able to image two fluorescent probes at up to 144x128x56 voxels at 30 volumes per second, allowing fast events to be followed in 3D in real time. In addition to the new detector technology, new excitation sources at 457 nm and 515 nm are essential to allow a range of different fluorescent probes to be used and significant modifications to the existing illumination system are required in order for multiple excitation wavelengths to be optimally employed. In summary, the proposed improvements to the OPM method will enable important sub-cellular mechanisms occurring during contractile events in cardiac myocytes to be studied quantitatively at high speed and in 3D for the first time.

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. OPM is implemented as '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 to sample preparation techniques currently used in 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 high-speed 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, in either a flow or imaging configuration. 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 and the preferred route for commercialization would be a licensing agreement with an established microscope manufacturer. Andor Technology has expressed significant interest in this technology and has written a strong letter of support for this application. However, the attractiveness of this technology to industry will be much greater once biological results are obtained using OPM, which is the key goal of this proposal. Such data could support a subsequent collaborative development programme, e.g. with Andor, that could be funded by a BBSRC Industrial Partnership Award or the Technology Strategy Board. Cardiac failure is an important public health problem with increasing prevalence. Independent of aetiology, the natural history of cardiac failure follows similar patterns. There are limited treatment options and a gradual decline in heart function occurs with a 5 year survival of only 50% once symptoms develop. The most important cause of mortality is sudden cardiac death, with 50% of heart failure patients dying suddenly. Ventricular tachyarrhythmias such as ventricular tachycardia and ventricular fibrillation account for the majority (60-80%) of these. With colleagues, KM has shown recently that in failing hearts Ca regulation is impaired and this occurs in parallel with ultrastructural changes. The impaired cellular Ca homeostasis can underlie the formation of cellular Ca waves which propagate slowly through the cells and result in depolarisations that may, in turn, trigger arrhythmias. Therefore there is a close relationship between cell structure, Ca homeostasis and arrythmogenicity. The novel approach to imaging detailed here will allow an understanding of the important sub-cellular mechanisms and a visualisation of these in 3D. Research directed at the cellular events will allow a more logical approach to the use of therapies. In the longer term OPM can benefit a wide range of different areas in biology which will impact basic biological research in a diverse range of areas, including studies of cell-signalling, immunology and embryology.