Grant: Research Tools and Instruments

Super resolution microscopy has broken the optical diffraction barrier enabling unprecedented imaging of 100 nm cellular and molecular structures. Resolution of single-molecule localization microscopy (SMLM), based on the repeated activation (blinking) of small numbers of discrete fluorophores, approaches 10 nm. Stimulated emission depletion (STED) super resolution microscopy, with a resolution of 40-50 nm in XY, is more versatile than SMLM and can image multiple fluorophores and live cells. At UBCs Life Sciences Institute (LSI), we have developed improved hardware (real-time drift control) and analytical approaches (machine learning network analysis and graph theory) for SMLM as well as 3-channel, 3D analysis using STED. Upgrading our capability with SMLM and STED is now critical. SMLM: Development of analytical tools to interpret the point distributions generated by SMLM is in its infancy. Collaborative efforts with a computer scientist (Hamarneh, SFU) and a mathematician (Coombs, UBC) led to the development of novel analytical approaches for cluster analysis of SMLM data. To validate these approaches and have hard measures of resolution, we will purchase Gattaquant DNA Origami. We will use these nm scale nanorulers (1D, 2D and 3D) to validate whether our cluster analysis tools report accurately on fluorescent probe localization and assess resolution limits of our SMLM microscopes. STED: We have applied 3-channel STED microscopy to define novel subdomain structure of ER tubules and to define the domains associated with tonic B cell receptor (BCR) signaling. To determine functional overlap between ER protein clusters and signaling molecules that associate with BCR clusters, we require a third 775 nm depletion laser to enable 4- and 5-channel STED. The 775 nm depletion laser reduces phototoxicity and enables 2-3-channel live cell imaging using infrared and far red probes. Upgrading our current Huygens STED analysis software is essential for multi-color 3D STED image processing and analysis. Development of our SMLM and STED capability is required for application of super resolution microscopy to multiple NSERC-funded research projects including: ribonucleoprotein stress granule complexes disassembly during RNA virus infection (Jan), membrane contact sites in yeast (Loewen), disk membrane evagination in photoreceptor cells (Moritz), gap junction remodeling in astrocytes (Naus), signaling during regeneration and degeneration of skeletal muscle (Rossi), cellulose synthase secretion and its impact on wood quality (Samuels), localization of calcium regulatory machinery in Sertoli cells (Vogl), and interaction of nano-biomaterials with osteoclasts and bacteria in bone fractures (Wang). LSI Imaging has over the past 3 years trained 299 users from 89 groups and our super-resolution expertise and technology will lead to enhanced HQP training in super resolution microscopy.