Multimodal Microscope for Live Cell Molecular Dynamics - Project Summary This proposal seeks to replace Colorado State University’s first open-access spinning disk confocal (SDC) microscope, installed in 2006, with a 3i Marianas super-resolution SDC, enhancing our research capabilities to meet the demands of modern biological imaging. Whereas a laser scanning microscope uses a single pinhole with the full intensity of the laser beam rastering across the object being imaged, an SDC spreads the laser beam over a rapidly spinning disk containing hundreds of pinholes and microlenses, returning the fluorescence emission through the same lenses, and building a full image much faster, often with less phototoxicity. Three major upgrades, internally funded by shared costs between users and a centrally funded core, have allowed our original SDC to function for 18 years, but its limitations for state-of-the art biological imaging in diverse model systems have become all too apparent. Our research utilizes a broad range of biological models such as yeast, mammalian cell culture, organoids, tissue slices, and model organisms including C. elegans and zebrafish. We require the ability to rapidly detect faint single-molecule fluorescence signals for studying dynamic processes such as transcription and translation, and many applications require broader fields of view, such as during oogenesis or following multiple cells in a field. The 3i Marianas microscope will meet these diverse and modern needs, providing our researchers with an easy-to-use super-resolution microscope that also provides a 3-fold improvement in imaging brightness, allowing for both improved imaging quality and reduced phototoxicity. The unique ability of the 3i Marianas microscope with the Vector3 TIRF module to rapidly switch between confocal and TIRF/HILO imaging modes will also give our researchers the ability to track single molecules while simultaneously co-imaging nearby structures with confocality, providing important biological context for acquired tracks, even in thick samples such as C. elegans or mammalian sperm. Finally, the ability to combine these imaging modalities with a photo-activation/conversion/photobleaching point scanner will enable the systematic measurement of subcellular protein dynamics, for example, the timing of receptor-ligand binding or the association of prion-like domains. Acquiring this microscope will significantly enhance our live-cell imaging capabilities, enabling unprecedented insights into cellular processes and expanding what is currently possible at our institution.
