Quantitative Optical Nanoscopy: Measuring the abundance and stoichiometry of proteins and nucleic acids with single-molecule microscopy

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The use of advanced optical imaging techniques to extract quantitative information on the abundance and stoichiometry of proteins or nucleic acids is termed optical proteomics' or optical genomics', respectively. Already, optical proteomics have been employed to probe a variety of complex protein networks, such as those involved in cancer formation. If molecular quantification or counting with conventional fluorescence microscopy is akin to an analog approach, the super-resolved technique single-molecule localization microscopy (SMLM) may provide a digital solution. A super-resolved, SMLM-based approach to molecular counting would be particularly powerful in single-cell, optical proteomics and genomics applications where trace amounts of protein, DNA, or RNA, must be detectedand often at spatial scales below the resolution limit of light microscopy. Single-molecule microscopy has the potential to significantly accelerate the optical -omics fields, leading to the development of new platforms for the characterization and identification of proteins and nucleic acids at ultra-low concentrations in vitro and in single-cells. Our group has developed both a theoretical foundation and experimental protocol for extracting accurate molecule counts from single-molecule localization (SMLM) microscopy data in the hopes that the technique becomes the future gold standard for counting molecules in cells. We have recently begun to apply our methods to address two important, outstanding biotechnical challenges, namely to: 1) measure the cell-to-cell variability in plasmid copy number and correlate that with protein expression (w/ McMillen Lab, Chemistry, U of T) and 2) quantify the membrane bound spatial organization of G protein-coupled receptors (GPCRs) and their associated G proteins (w/ Prosser Lab, Chemistry, U of T). These projects incorporate the training and mentoring of a large number of HQP in techniques at the boundary of the physical and life sciences, who will actively be recruited from a diverse pool of trainees. While currently confined to the research laboratory, as this technique matures, it could have a significant impact on industrial and biotechnological applications such as insulin production, drug-screening and medical diagnostics. However, this entire project is acutely dependent upon having scientific cameras that can rapidly detect light at the single photon level. Our current EMCCD cameras are approaching 10 years old, are at the end of their useful lifetime, and are significantly slower and less sensitive than modern, state-of-the art sCMOS cameras. This is severely impacting our ability to train HQP and to compete at an international level in this highly competitive fieldand will ultimately halt this research in the next few years unless the equipment is upgraded.