Probing cell and tissue dynamics with lightsheet microscopy

Imaging dynamic process in living samples is key to understanding biological regulatory mechanisms, whether at the level of molecules, cells, tissues or entire organisms. A major limitation however in most forms of microscopy is the sensitivity of living specimens to damage caused by the light used to view them, limiting the resolution, contrast and duration of time-lapse imaging. Such issues with existing live-imaging approaches have led in the past decade to the development of "light sheet" microscopy, based on optical principles developed over a century ago. These microscopes illuminate the sample with a thin sheet of light, allowing one see the sample in thin slices called 'optical sections'. By optically slicing the sample at different levels, one can build a three-dimensional view of the sample where the entire image is in focus. Other existing technologies such as confocal microscopy can create such 'optical section', but due to the way they work, impose much greater light induced damage to the sample than light sheet microscopes. A further advantage of light sheet microscopes is that the sample can be imaged from different angles. Images from these different views can then be combined to give one a very high quality 3-D representation of the sample.

This revolutionary new technology has only become commercially available in the end of 2012, from Zeiss. We tested the Zeiss microscope during the two weeks it was left here in Oxford on demo and found it to be extremely well engineered and user friendly. After less than a day of training, users were able to use it entirely independently. Because of their unique architecture, one of the most challenging aspects of light sheet microscopes is sample mounting. The instrument form Zeiss was particularly impressive in this respect, allowing one to easily mount and locate the sample, as well as maintain samples under very stable conditions suitable for extended culture and imaging, making it ideal for the projects described in this proposal.

We propose to purchase a Zeiss Lightsheet Z.1 microscope for the use of the research community in Oxford and surrounding areas such as Harwell and Bristol. The microscope will be managed by and take advantage of existing expertise in Micron Oxford, an advanced bioimaging unit that has rappidly established itself as a centralised facility serving the imaging needs of several departments in the University of Oxford.

The applicants will use the Zeiss Z.1 to address: basic biological problems that underpin future developments in human health, such as how cells in the body divide or move from one place to another; how memories are established, which will inform our understanding of the cognitive decline that accompanies aging; how stem cells produce the cells in our blood and; how the heart is built and responds to injuries, which will contribute significantly to our capabilities in regenerative medicine.

The grant would be used for the purchase of a Zeiss Lightsheet Z.1 microscope. Like confocal microscopes, Lightsheet microscopes provide optical sections, but have several additional advantages because the architecture of the instrument is completely different. They cause much less damage to the sample (both photothermal and photochemical) so are ideal for time-lapse imaging of live samples. They can also easily image the sample from multiple angles, so one can build a tomographic image. This allows one to image larger samples than with a standard confocal, without compromising on resolution, which is comparable to that of wide-field systems in the image plane, and higher than conventional systems in the axial plane (the different z levels). Finally, they are very fast, collecting at over 30 frames per second.

The Zeiss Z.1 is the first commercially available light sheet microscope and has generated a great deal of excitement in the imaging community. We tested the Z.1 extensively and its capabilities in live cell imaging and fluorescence tomography compared with a leading wide field instrument (DeltaVIsion from GE), a leading spinning disc microscope (Ultraview from Perkin Elmer) and confocal laser scanning instruments from Zeiss, Leica and Olympus. The Z.1 has clear advantages for a number of key experiments in living cells, both in terms of maintaining sample viability, since the light sheet causes considerably less bleaching and photo damage than all the other modalities, but also in its ability to carry out tomography on intact tissues. We would use the Z.1 to address basic biological problems that underpin future developments in human health, are relevant to our understanding of capabilities such as memory, that deteriorate with age and will drive forward our understanding of stem cell biology and capabilities in regenerative medicine.

Acquisition of the Zeiss Lightsheet Z.1 microscope will advance imaging nationally and internationally, by leveraging the existing expertise at and collaborative environment within Oxford University. By managing access to this microscope through existing mechanisms developed by Micron Oxford, a centralised bioimaging unit, the microscope will be made widely available to the research community not only in Oxford, but also outside, for example the University of Bristol and MRC Harwell, fostering an environment that encourages wide-ranging multi-disciplinary interactions. This strategic vision for the future will continue to keep Oxford at the international forefront of this increasingly exciting phase of biomedical basic and translational research.

Our use of the light sheet microscope are will result in basic discoveries thats can be translated into clinical applications. This will, in the long term, contribute to benefits in human wellbeing and to the economy. Our research will therefore have an impact on the general public and commercial sector. Given the medical relevance and ethical implications of understanding developmental processes, and its impact on stem cell biology, our work will provide factual input into and therefore benefit the public discussion about the advantages and risks of stem cell therapy.

The results of this research will be conveyed to other researchers through the publication of findings in peer-reviewed journals, by reporting unpublished work at conferences and through personal communication with other scientists. Though the results will primarily be disseminated through scientific journals, attempts will be made to inform the media of results prior to publication, so that the general public is more likely to be made aware of the results. We take seriously the responsibility of scientists to engage with the lay public, to raise awareness among them of the results of publicly funded research, to openly debate ethical issues relating to our research so that public opinion may be formed in an informed manner and to take the excitement of our research to the children of today, who will be the scientists of tomorrow. For these reasons we engage through the university but also as individuals in activities aimed at the public dissemination of science.

A dedicated web page for Micron already exists, that serves not only users, by providing a space for technical information on sample preparation for microscopy etc. but also contains pages in plain english, to promote advanced imaging approaches such as light sheet microscope to the wider community beyond researchers.

This project will also train a young researcher in emerging methodologies, contributing to their career development, as well as producing an individual capable of carrying on future research in the biomedical sciences. Micron runs an annual microscopy course that has now been over subscribed for the third consecutive year. This course is taught by several of the participants on this bid and covers a variety of imaging modalities and will include a session on light sheet microscopy.

We have set up a Bioimaging Strategy Group (BSG) comprising leading academics involved in imaging research from all interested Departments within the University. This group is chaired by Jordan Raff, one of the investigators on this application. The BSG has a central role in driving optimal coordination of research into high-end microscope development and use in Oxford. It helps prioritise and make plans for future developments and specific bids for microscopy infrastructure. The impact of the light sheet microscope will be enhanced by our coordinated plans for data storage, retrieval and fast transfer between sites, which is likely to continually increase.