Lattice Selective Plane Illumination Microscopy (L-SPIM) for the analysis of subcellular dynamics in living specimens.

The discovery of the green fluorescent protein from the jellyfish Aequorea victoria has revolutionised our way in which we study cells in a tissue but also our approaches to investigate signalling events, organelle dynamics and interactions of organelles within the complex environment of the living cell. Parallel to the developments in GFP biology, there have been advances in fluorescence imaging methods and microscopical systems that make it possible to follow fluorescently labelled cells, organelles and cytoskeleton elements, to quantify their abundance and to probe their mobility and interactions. However, long-term imaging of complex 3D tissues with conventional laser-scanning microscopes is still one of the most significant obstacles in fluorescent microscopy as this microscopy induces phototoxicity and photobleaching. Selective plane illumination microscopes (SPIM) has been developed allowing long-term imaging by scanning the specimen with much less laser-induced damage such as phototoxicity and photobleaching. However, subcellular structures deep in tissue are still a challenge to resolve. Due to the recent development of Lattice SPIM, it is now possible to image small structures such as vesicles or microtubules over an extended scanning time deep in tissue without damaging the specimen. The combined advances in GFP biology and imaging methods are providing a massive opportunity for investigating the kinetic properties of organelles in living cells.

The University of Exeter studies many aspects of biological and biomedical research, reaching from fungal-related plant disease research to signalling biology in vertebrate embryonic development. In the past, microscopical approaches such as confocal microscopy and electron microscopy have been used to investigate subcellular interactions. These observations can now be complemented and extended in real-time in living cells in complex organisms. The latest generation of Lattice SPIM is, therefore, a game-changer for cell biology - in bacteria, fungi, plants and animals. It is now possible to generate dynamic maps of organelles in living cells using a fluorescence microscope over a timescale of hours to days, which has not previously been possible. In this application, we seek support to purchase a modern Lattice SPIM, which will complement the existing facilities at the Exeter Bioimaging Centre.

The development of the many different genetically encoded fluorescent proteins has sparked a revolution in optical imaging in biological and biomedical research. These fluorescent proteins have expanded the repertoire of imaging applications from multi-colour imaging of protein co-localization and organelle dynamics to the detection of changes in intracellular activities, such as pH or ion concentration of living cells. A game-changing technology has taken centre stage to analyse these biological processes, the lattice selective plane illumination microscopy.

First developed by Nobel Laureate Eric Betzig, Lattice Selective Plane Illumination microscopy (L-SPIM) is capable of imaging biological systems spanning four orders of magnitude in space and time. L-SPIM generates an optical lattice to create an ultra-thin light sheet to image biological samples over long periods of time and with very fine resolution. Conventional fluorescent imaging experiments are limited to seconds or minutes, however, the imaging on an L-SPIM can be extended to hours or even days. The combination of high spatiotemporal resolution, imaging speed and sensitivity make L-SPIM the ultimate imaging tool for a new era of living cell microscopy.
This application seeks funds to purchase a modern L-SPIM
(a) to investigate the molecular dynamics of organelles in the cell in the living, complex organism and
(b) to measure signalling events based on fluorescent reporter systems in real-time in the living organism.

The new instrument will be housed and integrated into the existing University of Exeter Bioimaging Centre, thereby adding additional capabilities. As such, it will be accessible to additional users and, therefore, will significantly improve the local research infrastructure at Exeter as well as in the South West of England including the GW4 universities Bristol, Bath and Cardiff.

The gene encoding green fluorescent protein (GFP) has recently become an important tool to visualize cellular organelles in unicellular and multicellular organisms. In 2008, the Nobel Prize in Chemistry has been awarded jointly to Japanese scientist Osamu Shimomura and US researchers Martin Chalfie and Roger Tsien, for the discovery and development of the GFP. Parallel to the developments in GFP biology, there have been advances in imaging technologies that make it possible to localize proteins fused with GFP by sensitive imaging technologies. And indeed, six years later the Nobel Prize in Chemistry was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner for the development of super-resolved fluorescence microscopy. In light of these developments, imaging of biological samples has become an area of outstanding interest for BBSRC. Bioimaging technologies cut across all areas of BBSRC's remit, including plant, fungal and animal biology. Technology development has been rapid; with advances in sensitivity, resolution, speed and signal processing; allowing researchers to visualise and measure biological processes in complex biological tissues.
We base our application on these fundamental discoveries. The impact of our proposed work is therefore timely and significant on the world's stage with regard to dynamic biological processes of sub-cellular structures cross-species. Our work will draw together the disciplines of cell and developmental biology, plant biology, fungal disease biology, ecosystem health, cell movement dynamics and effector biology.

Our work will impact upon
i) Knowledge - giving a greater understanding of the organelle dynamics and signalling processes in living cells
ii) UK science - enhancing the profile of the Investigators on the national and international stage
iii) Interdisciplinary science - endowing the Investigators with new awareness and widened interdisciplinary skills
iv) Fostering broadened industrial links with microscopy companies such as Luxendo/Bruker but also pharmaceutical companies such as AstraZeneca and Syngenta.
v) Fuelling greater research effort across the GW4 universities.
vi) Enhancing skills of the experimental officer (EO) and training new staff and students
vii) Promoting public awareness of science and raising awareness of the importance of fluorescence imaging technologies in life science.