Application for a TRI-SPIM fluorescence lightsheet microscope

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

This is an application for a high resolution TRI-SPIM light sheet fluorescence microscope (LSFM). This cutting edge LSFM will enable the acquisition of 3D data stacks at high spatial and temporal resolution from living samples with minimal photodamage. The TRI-SPIM will greatly enhance Dundee's study of key biological processes and be first of its kind in the UK. The TRI-SPIM uses alternating dual lightsheet illumination of samples mounted horizontally on a transparent substrate, while collecting simultaneous images from a third side, making optimal use of all the information available. This LSFM will allow much needed, improved spatio-temporal resolution during live imaging of critical processes from the subcellular to the cellular and tissue scale. This technology will be of immediate use in addressing key questions on the mechanism of mitosis, including formation of the microtubule spindle formation in IPS and ES cells, the mechanism of microtubule kinetochore attachment during chromosome bi-orientation and error correction in yeast and mammalian cells, the separation of daughter chromosomes in C elegans and the role of Ste20 kinases in linking tumour suppressors to spindle orientation and anchoring in normal and cancer cells. The advanced imaging capabilities will be used to investigate the molecular mechanisms underlying the establishment of polarity during the asymmetric division of neural stem cells as well as the molecular mechanisms of apical abscission essential for ingression of neuro-epithelial cells during their differentiation in the formation of spinal cord and ingression of mesendoderm cells during gastrulation. The microscope will be essential for imaging the 3D dynamics of the actin-myosin cytoskeleton during chemotactic movement in Dictyostelium and vertebrate mesendoderm cells. It will also allow further development of advanced LSFM capabilities and present a major new development in the Dundee Imaging Facility and the greater UK community

The establishment of high resolution lightsheet based live imaging techniques will benefit many internal and external academic beneficiaries as indicated in the relevant section, through access through the microscope and its advanced imaging capabilities. These will allow them to push forward their research in new directions. It will also be instrumental in the training of new researchers such as PhD students and Postdocs in advanced live imaging and data processing techniques.

Through the expected novel insights in key areas of key life science research areas it will benefit the wider biology and biomedical and biotechnology research community. For instance increased understanding of the control of asymmetric divisions of stem cells will be critical for the development of the rational use of stem cells in regenerative medicine. Novel insights in EMT and cell migration will be critical to understanding diseases such as the immune system and cancer. Findings made suing these in-vivo imaging techniques will be critical to the development and validation of novel biomarkers, development and validation of drug targets, especially relevant to the biotech and pharmaceutical sector.

The project will also help to drive the development of this new light sheet technology forward not only in imaging and sample preparation methodology, but also in image data processing and analysis. The incorporation of in vivo manipulation techniques such as optogenetics, microsurgery and optical manipulation through tweezing will further expand the range of beneficiaries of this technology. All these activities will drive and impact further interdisciplinary collaborations with physicists, engineers and computer scientists both in the academic and private sector to mutual benefit.