The parent Focused Technology RO1 proposes to develop universal, easy-to-use methods that will enable any super-resolution microscopy user to quantify the stoichiometry of proteins in super-resolution images. Super-resolution microscopy has revolutionized how we visualize the inner life of cells. Despite its impact, super-resolution microcopy currently suffers from important limitations. Specifically, counting proteins in super-resolution images is extremely challenging. The parent RO1 will develop and validate new methods to overcome this major challenge. As such, the project heavily relies on the use of specialized super-resolution microscopes capable of single molecule detection. Over the years we have tested several commercial super- resolution microscope systems and identified the Nanoimager from Oxford Nanoimaging (ONI) to have several favorable capabilities: easy-to-use user interface, compact footprint and mechanical stability that does not require the use of an optical table. We currently own an old, beta-version of the Nanoimager, which has several limitations typical of beta-testing systems. Importantly, the beta-version is not robust. Indeed, since its initial purchase, we had several instances of major failure putting the microscope out of commission for periods of more than 6 months. In addition, the Nanoimager is in heavy demand with 10 heavy users (>10 hours per week per user) and 3 medium users (~5 hours per week per user). Importantly, super-resolution microscopy is a fast evolving field and since the development of the beta-system, ONI has drastically improved the robust operation of their microscope as well as added new hardware/software features for high throughput, automated imaging as well as 3D imaging capabilities, which are important for but currently not available to the parent RO1 project. Therefore, the parent RO1 is jeopardized by relying on an outdated, non-robust and heavily used microscope. Accordingly, we request funds to purchase the most up-to-date version of the Nanoimager to be used exclusively for parent RO1 projects.
This proposal will develop standardized methods and software that are easily accessible to non-expert users of single molecule super-resolution microscopy and that will empower them with quantitative tools to measure protein stoichiometry with nanoscale spatial resolution. We will develop methods based on calibration nanotemplates and the novel concept of benchmarking standards, which will enable quantification of copy number of small and large multi-protein complexes independently of experimental conditions. We will further implement an innovative, user-friendly, open-source, all-integrated software that incorporates all the steps needed to determine copy number distribution of proteins in super-resolution images.