An Enhanced Combinatorial Super-Resolution Microscopy Platform

RTI资金将用于提高超分辨率组合显微镜平台的空间、时间和光谱分辨率，该平台将扫描探针显微镜的亚纳米3-D分辨率与超分辨率荧光显微镜相结合。后一种技术(及其变种)最近被授予2014年诺贝尔奖。即将升级的平台在加拿大是独一无二的，缺乏商业系统的能力。该平台包括已有15年历史的扫描探针显微镜(SPM)、自制的自费4激光双相机全内反射显微镜和4激光共焦显微镜，对于多个成功的跨学科、教职员工和大学合作至关重要，广泛支持结构生物学、生物物理学和工程学项目。本提案寻求资金，用一种最先进的系统取代目前的扫描显微镜，该系统具有增强的扫描性能、显著降低的噪音以及更高的灵敏度和分辨率。由于供应商不再支持当前的SPM，并且已变得越来越不可靠，因此迫切需要这种替换。零部件不能采购，因为它们不再是制造的，这使得eBay和其他拍卖网站成为零部件的唯一替代品。我们还在寻求资金，以便将561 nm激光整合到现有的光学系列(405 nm/473 nm/532 nm/637 nm激光线路)中。这条线将激发许多用于超分辨率成像的关键荧光团。我们光学系列的开束设计允许随时控制相位、对准、偏振和光束形状，这些属性在商业系统中不是那么容易操纵的。

RTI funding will be used to enhance the spatial, temporal, and spectral resolution of an exceptionally capable super-resolution combinatorial microscopy platform that combines the sub-nanometer 3-D resolution of scanning probe microscopy with super-resolution fluorescence microscopy. This latter technique (and its variants) was recently awarded the 2014 Nobel Prize. The platform to be upgraded is unique in Canada with capabilities lacking in commercial systems. Comprising a 15 year old scanning probe microscope (SPM), a home-built, self-funded 4-laser dual-camera total internal reflection microscope, and a 4-laser confocal microscope, this platform is critical to multiple successful inter-disciplinary, -faculty, and -university collaborations, broadly supporting projects in structural biology, biophysics, and engineering. The present proposal seeks funds to replace the current SPM with a state-of-the-art system that has enhanced scan performance, significantly reduced noise, and higher sensitivity and resolution. This replacement is critically required as the current SPM is no longer supported by the vendor, and has become increasingly unreliable. Components cannot be sourced as they are no longer being manufactured leaving eBay and other auction sites as the only alternatives for parts. We are also seeking funding for a 561 nm laser to be integrated into the existing optical train (405 nm / 473 nm / 532 nm / 637 nm laser lines). This line will excite many of the key fluorophores for super-resolution imaging. The open-beam design of our optical train allows for ready control over phase, alignment, polarization, and beam shape, attributes that are not as easily manipulated within commercial systems.