Tuesday, February 4, 2025
2/4/2025
7. Project Summary/Abstract Fluorescence microscopy is one of the most powerful and versatile techniques available for biological studies. These days, fluorophore-labeled molecules and genetically encoded fluorescent proteins are often bright and readily distinguishable from background signals, making it easy to obtain high contrast images and measure protein expression, localization, and activity in living cells. However, in light microscopy, resolution is fundamentally limited by the properties of light diffraction, which prevents the resolution of structures smaller than approximately half the wavelength of light. Electron microscopy has a much higher resolution than light microscopy and has long been relied on to visualize cellular structures smaller and/or closer together than 250 nm. However, fixation, dehydration, and ultrathin sectioning are required during sample preparation for electron microscopy, making it technically challenging, prone to artefacts, and incompatible with live imaging. Therefore, microscopic techniques that combine the nondestructive nature of light microscopy and the nanometer resolution of electron microscopy (i.e., super-resolution techniques) have been the focus of much research and development in recent years. We propose to purchase an Elyra 7 Lattice SIM2 system, which achieves substantial improvements in spatial and temporal resolution and efficiency while decreasing the light dosage to the sample in two ways. The first way involves the use of lattice structured illumination microscopy (SIM). In lattice SIM, the sample area is illuminated with a lattice spot pattern, which leads to a dramatic increase in imaging speed, higher contrast, more robust image reconstruction, and less laser dosage for sample illumination than conventional SIM. The second way involves the use of a novel image reconstruction algorithm, termed dual iterative SIM or SIM2. At Loyola University Chicago, the NIH sponsored research of many scientist requires fluorescent microscopy to characterize the interaction between small structures (e.g. virus, proteins, RNA) and organelles that mediate key biological functions. To understand these interactions, is critical that our scientists have the ability to resolve them with the finest possible detail and to characterize how cellular and molecular events unfold in real time in a live context. The Elyra 7 Lattice SIM2 system will be housed in our Core Imaging Facility, giving widespread access to this revolutionary imaging technology. Compared to similar instruments in the Chicago area, this instrument boasts unmatched versatility, allows for superior temporal and spatial resolution, is more efficient, and has a larger field of view. Therefore, this system will greatly enhance the NIH sponsored research of the Loyola University Chicago user group.
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