A Turn-Key Single Molecule Localization Microscope for Imaging Extracellular Vesicles and Nanoparticles

SUMMARY We request funds to purchase an ONI Nanoimager super-resolution microscope to support cutting-edge NIH- funded extracellular vesicle (EV) and nanoparticle research at Vanderbilt University (VU) and Vanderbilt University Medical Center (VUMC). The ONI Nanoimager is a class 1 laser, four-color fluorescence super- resolution microscope that provides a user-friendly solution for resolving nanoscale structures at 20 nm resolution. Its patented vibration-dampening microscope body can be placed on a standard lab desktop and yet generate single molecule localization data. The Nanoimager was developed with state-of-the-art characterization of EVs, nanoparticles, and other nanoscale structures in mind. It performs single-molecule localization microscopy (SMLM) modalities, including dSTORM, PALM, and PAINT, and is uniquely capable of super-resolution single-molecule FRET and single particle tracking. It further surpasses the function of other shared super-resolution microscopes at Vanderbilt by performing simultaneous two channel imaging and has a superior 100X magnification,1.45 numerical aperture objective. The light engine is equipped with 405 nm, 488 nm, 560 nm, and 640 nm high-powered lasers and a dichroic mirror split at 640 nm. Users can adjust the illumination angle in 0.5 degree increments for imaging in epifluorescence, HILO, and TIRF modes. The accessory environmental chamber allows extended live cell imaging under physiologically supportive conditions, while the APLO Flow microfluidic device provides our EV research users with end-to-end automation, from EV capture to labeling to imaging, and the capacity to perform microfluidics-based experiments. Operation of the Nanoimager is remarkably simple, with many customizable pre-built imaging protocols and powerful data analysis tools available. These attributes are quite unusual for a super-resolution microscope, making it ideally suited as a Core instrument. Our Major and Minor Users will use the Nanoimager’s super-resolution imaging capabilities to study diverse biological processes in cell and cancer biology, including immunology, neurobiology, drug delivery design, and biomarker studies. Major users' projects focus on EV and nanoparticle biogenesis and composition (Pua, Weaver, Coffey, Wilson), disease biomarkers (DelGiorno), EV functions in extracellular matrix organization (Weaver), and EV and nanoparticle immunopotentiation and suppression (Coffey, Wilson). The Vanderbilt Cell Imaging Shared Resource (CISR) and the Vanderbilt Center for EV Research will promote the utility of the Nanoimager to VU and VUMC researchers through seminars, workshops, and website content. CISR will administrate scheduling, train users in the operation of the instrument, manage use, and apply an established business model to support the long- term maintenance of the system. In summary, the ONI Nanoimager will permit multiple modes of high-speed super-resolution imaging and single particle analysis designed to support cutting-edge EV and nanoparticle research not currently available to the Vanderbilt community in a compact and user-friendly format.