A multi-user confocal superresolution microscope for cell and developmental biology

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Developments in microscopy have catalyzed ever more sophisticated understanding of how cells and organisms work. Fluorescence microscopy, allows us to monitor individual proteins, protein complexes, or organelles, using antibodies or protein tagging, in either fixed or live preparations; this in turn helps us link the behavior of individual proteins or protein complexes with molecular and genetic characterization of their properties.However, normal microscopes cannot distinguish between objects that are separated by less than half the wavelength of visible light, or around 200-300 nanometers (millionths of a millimeter). Since many components of the cell machinery are separated by distances below this limit, understanding how this machinery works requires microscopy techniques that can resolve even below this fundamental limit. This compelling need has driven the recent development of a number of "super-resolution" microscopy approaches by (among others) the 2014 Chemistry Nobel Laureates Betzig, Hell and Moerner. Each of these approaches has its own strengths and limitations. We are applying for one type of super-resolution microscope, called a Stimulated Emission Depletion (STED) microscope. In this, laser beams use properties of fluorescent labels to generate "pixels" of light from the preparation that are smaller than the 200-300 nanometer limit - typically 50 nanometers or less, which is sometimes enough to distinguish even the opposite ends of the same protein molecule. We have chosen STED because it meets the needs of a wide range of users in cell and developmental biology: it allows super-resolution in all three dimensions, is fast enough to image live preparations in real time, and allows us to image at greater depths into preparations than other super-resolution methods.We will set up this microscope in an environment that permits its use by as wide a range of users as possible. STED is now mature enough that we can procure a commercially available instrument that is sufficiently well configured and supported to allow use by trained non-specialists. Wide use will be facilitated by specialised technical support, a management committee, a web-based booking system, extensive documentation, user training, and a program of education for users.We already have an active user community who need super-resolution microscopy to understand the molecular basis of fundamental cellular processes in a range of model organisms including yeast, fruitflies, and human cells. These processes represent the core biological functions of the cell, all of which can go awry in a large variety of of long term health problems that include neurological diseases and cancer. Our findings will therefore underpin our knowledge of these diseases. Examples of how a STED microscope will help advance these projects are:1. To localize membrane structures within neurons, especially in the confined spaces of axons and synapses where signals are transmitted; and detecting defects in these structures in fruitflies carrying mutations homologous to human axon degeneration mutations. 2. To understand how neuronal networks form by studying the mechanisms that regulate the development and maintenance of synaptic contacts between identified neurons. 3. To understand the organization and localization of multifunctional cellular machines that can choreograph cell division to achieve equal segregation of cell components into two daughter cells, and that can also organise cilia, cellular outgrowths that can have both motor functions and also act as signalling antennae. 4. To understand mechanisms that achieve unequal segregation of proteins and RNAs during cell division, to ensure correct differentiation of different cell types. 5. To assess overlap and cooperativity in the functions of nuclear proteins through studies of their precise three-dimensional organization to regulate gene expression.