MICA: High speed, high resolution imaging of excitable cell networks

The heart and brain function through the fast propagation of electrical impulses. Heart arrhythmias and epileptic seizures are the results of massive electrical surges in these organs, but all cardiac and neurological dysfunction results ultimately from alterations in the patterns of biological electrical activity.

Visualising activity in cardiac and neuronal tissue is a fundamental challenge for biomedicine, as each brief electrical signal propagates rapidly over large distances. For example, a single heartbeat involves choreographed waves of electrical activity pervading every cell in the organ. During the same time period, electrical signals in the nervous system can be used to detect the light reflected from an incoming cricket ball, calculate its trajectory, and send the motor commands to make the catch. Our proposal brings together two new techniques in optical engineering that will enable us to capture these rapid events in electrically excitable tissues.

This proposal will create a high speed imaging facility, which will enable the wider application of these cutting-edge approaches to studying cardiac and neurological disease.

Advances in biomedicine have always been paced by developments in the physical and engineering sciences, which enable us to peer ever deeper into the nature's intricate mechanisms. We are now at another watershed, with cutting-edge techniques in optical microscopy offering the opportunity to resolve the dynamic behaviour of excitable cell networks, with a combination of high speed 3D scanning and subcellular spatial resolution. The aim of this proposal is to capitalise on these UK innovations in optical microscopy, and establish the technologies in a life sciences department in which they can be applied rapidly to biomedical questions. The imaging facility will implement turn-key solutions for three synergistic technologies:

a) Remote focusing two-photon microscopy, which enables scanning at kHz rates along arbitrary 3D trajectories in thick biological specimens, and thus the imaging of activity propagation within and between excitable cells.
b) High speed (1000 fps) confocal microscopy, using structured light and a temporal pixel multiplexing camera, which enables simultaneous high speed imaging of cellular activity and high spatial resolution imaging of anatomical structure.
c) Multi-focal structured illumination microscopy (mSIM), which enables high speed imaging at spatial resolution below the diffraction limit.

These high speed microscopy techniques offer various trade-offs in temporal and spatial resolution, and will be implemented on the same microscope to offer the most flexible imaging system. The hardware and software implementations will be honed iteratively through application to cardiac and neuronal preparations, both in vitro and in vivo, yielding a sustainable imaging system for studying excitable cell physiology and pathology.

Our objective is to establish an imaging facility that harnesses UK innovations in optical engineering, to promote cutting-edge UK biomedical research. This has the potential to impact our national competiveness at multiple levels:

1) Optical Engineering
Prototype bays within the imaging facility and IBIOS, in addition to pump-priming grants for equipment development, will allow continued development of the microscopy techniques. This can be used to take advantage of the high powered laser sources or develop bolt-ons for existing technology, thus ensuring continued engineering innovation.
The facility will also expose optical and software engineers to biomedical problems, which will inform equipment design and implementation. Moreover, these interactions will provide essential skills that can be applied to biomedical engineering problems in both academia and industry.

2) Biomedical Science
Establishing the techniques for high speed deep tissue imaging within the Department of Physiology, Anatomy & Genetics, Oxford, will provide the surrounding infrastructure to support biomedical applications in vitro and in vivo. An online booking system will enable local access to the technology for new biomedical applications, with pump-priming grants to foster wider usage, and thereby promote this cutting-edge optical microscopy across the UK.

3) Industrial Partnership
Our industrial partner, Cordin Scientific Imaging, will contribute to establishing the high speed imaging facility through development and manufacture of the TPM image sensor. The benefit for the industrial partner is that they will be able to test and tailor the components for biomedical applications, and showcase their applications for future end users.

4) Commercial Sector
Since the inception of stimulated emission depletion (STED) microscopy by Stephen Hell in 1986, this technique has been applied rapidly to biological questions, thereby promoting it as a useful resource. The STED microscopy continues to develop, but is now offered as a turn-key solution by Leica Microsystems, making it available worldwide. This emphasises the importance of close collaboration between engineering and biomedical scientists in driving innovation.
Our approach to high speed imaging will use a newly developed remote-focusing two-photon microscope and temporal pixel multiplexing, both developed in Oxford. The remote focussing technology has been patented via Isis Innovation, the University of Oxford's technology transfer company, with the patent currently under license to Zeiss GmbH. The temporal pixel multiplexing camera technology is under technology transfer by Isis Innovation, and being developed in partnership with Cordin Scientific Imaging. Further experience in technology commercialisation comes from the lead PI who personally has authored 12 patents (several of which are under licence) and founded two spin-out companies. The results of the research will highlight the potential of these techniques, and thus enhance the prospects for further commercialisation of the microscope system, which would enable its future application to a wider range of bioscience problems. Such technology transfer is a key to continuing advances in biomedical science in the UK and abroad.

5) Public
The public are potential indirect beneficiaries of this research, through advances in the understanding of the biological processes underlying health and disease, driven by developments in optical microscopy.