Application for a TRI-SPIM fluorescence lightsheet microscope

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An important goal in Life Science research is to study and understand the complex spatio-temporal dynamics of key processes that underlie living systems at all levels of organisation. Studying how various molecules interact to form organelles such as membranes, the cytoskeleton, the nucleus, the Golgi apparatus, lysosomes and energy producing mitochondria is a major goal of biochemistry, cell biology and molecular genetics. Understanding how cells perform different complex functions such a self-replication, shape change, movement and differentiation to form tissues, organs and even organisms are other key goals of cell and developmental biology and genetics and have many important implications for our understanding of organisms in health and disease. The frontier of these techniques is to study the full spectrum of complex dynamics from the molecular, cellular, tissue to organism scale in living systems at the highest possible spatio-temporal resolution. Light microscopy based imaging is a key methodology provides the ability to perform quantitative measurements molecular, cellular and tissue dynamics in living systems. Fluorescence light microscopy is a key merging technique of choice to study processes at the molecular, cellular, tissue and even small organism scale. Fluorescence based imaging is very powerful since it allows, detection of very specific labelled components with high specificity and contrast. Furthermore it has become possible to label many molecules specifically in-vivo with fluorescent proteins. In general there is a need to minimise the irradiation with light, since exposure to many high energy photons results in damage to the molecules and systems to be studied. Ideally the light intensity used should not exceed more than that of the equivalent of one sun in the sky. A recently developed technique known as light sheet microscopy goes a long way in reaching these goals, excellent spatiotemporal resolution during imaging of live samples while greatly reducing the radiation load due to selective illumination of only the part of the sample that is being imaged. This is achieved by separating the illumination path from the imaging path, which are typically at 90 degrees to each other. The lightsheet generates a very thin sheet of light that bisects a specimen and a second objective is used to image this illuminated section on a high sensitivity and resolution camera.The instrument, a triSPIM lightsheet microscope that we aim to acquire in this project improves this technology by using illumination from two opposing directions and simultaneous light collection from three sides. This results in optimal collection of the fluorescence signals and an improved resolution. This instruments will put our live imaging capabilities at the forefront of what is presently technically possible. A group experienced researchers will use this to study the mechanism underlying cell replication, including formation of the mitotic spindle, line up of chromosomes on the metaphase plate in dividing cells, chromosome separation. Further studies are aimed at understanding the mechanisms of cell polarisation and asymmetric division of neural precursor cells during formation of the spinal cord and brain as well as the role of stem cells in the function of the gut and the role of cell shape changes and cell motility, important during embryonic development and the function of the immune system. These and other future research projects to be tackled will have important consequences for our understanding of health and disease. As soon as this instrument is well established it will without doubt be used in many other cutting edge research projects. At present this will be the first microscope of its kind in the UK. Finally we plan to use this instrument as a basis to drive forward the development of this type of lightsheet microscopy based imaging technology of critical processes in living systems