A lattice lightsheet microscope for imaging highly dynamic processes in living cells and organisms.

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Light microscopy lies at the heart of biological research. Ever since the light microscope was invented in the seventeenth century, it has been revealing profound insights into how cells function, grow, divide and die, and how individual cells work together to generate tissues and organisms. The development of fluorescent probes for studying protein function in living cells coupled with extraordinary progress in microscope design have signalled an era of unprecedented insight into cell function via light microscopy. This has been marked by two recent Nobel Prizes. One was awarded in 2008 for the discovery of green fluorescent protein from jelly fish, which can be joined to a protein of interest by genetic engineering. The chimeric protein can then be expressed and observed in living cells. Since then, proteins that fluoresce in different colours have been identified, allowing researchers to follow multiple proteins in the same cell at the same time, providing vital information about cell behaviour. The second Nobel Prize was awarded in 2014 for the development of super-resolution microscopy. These methods gave a way of seeing structures with great level of detail than possible using diffraction-limited techniques, where the resolution is set by Ernst Abbe's nineteenth century equation.One of the 2014 winners, Eric Betzig, has gone on to design a microscope called the lattice lightsheet microscope (LLSM). This technological breakthrough has many advantages. It captures images in 3D very rapidly, meaning that cellular structures that move very fast inside the cell can now be followed in 3D. For slower-moving structures, it offers the option of super-resolution imaging within living cells. Lastly, and perhaps most importantly, the lattice light-sheet illumination is very gentle on cells, tissues and organisms, because it causes minimal photo-damage. This means that cellular processes can be followed for longer times than previously possible, and at faster speeds and higher resolution.This ground-breaking technology is now commercially available from Intelligent Imaging Innovations (3i), and here we propose to use the LLSM to image a wide range of different cellular structures within living cells over timescales ranging from minutes to days. We will be able to analyse the behaviour of fast-moving components such as endosomes and mRNA particles, and the cargoes transported by fast axonal transport in nerve cells. We will also image structural components of the cell's cytoskeleton - actin filaments and microtubules - as they work in processes as varied as cell division, migration and cell-cell communication. The LLSM's ability to image samples of different thickness will allow us to follow these processes in samples ranging from single cells through to cells in tissue samples or 3D cultures. In addition, we can use it to watch cell behaviour in developing embryos of fruit fly and zebrafish. The LLSM will benefit at least 24 groups of highly productive scientists holding significant BBSRC funding. This will also enhance the training of the next generation of researchers in a sophisticated light microscopic technique and the data analysis needed to interpret the results. In addition, the quantitative data generated will be used enhance collaborations between biologists, mathematicians and computer scientists, so promoting interdisciplinary research.