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

使用先进的光学成像技术来提取关于蛋白质或核酸丰度和化学计量的定量信息分别被称为光学蛋白质组学或光学基因组学。光学蛋白质组学已经被用于探索各种复杂的蛋白质网络，例如那些参与癌症形成的蛋白质网络。如果用常规荧光显微镜进行分子定量或计数类似于一种模拟方法，那么超分辨技术单分子定位显微镜(SMLM)可能提供一种数字解决方案。 一种基于SMLM的超分辨分子计数方法在单细胞、光学蛋白质组学和基因组学应用中将特别强大，在这些应用中，必须检测到微量的蛋白质、DNA或RNA，并且通常是在低于光学显微镜分辨率极限的空间尺度上。单分子显微镜有可能极大地加速光学组学领域的发展，从而为体外和单细胞中超低浓度的蛋白质和核酸的表征和鉴定提供新的平台。 我们团队开发了从单分子定位(SMLM)显微镜数据中提取准确的分子计数的理论基础和实验方案，希望该技术成为未来细胞中分子计数的金标准。我们最近已经开始应用我们的方法来解决两个重要的、突出的生物技术挑战，即：1)测量质粒拷贝数的细胞间变异性并将其与蛋白质表达相关联(w/McMillen Lab，Chemical，U of T)和2)定量G蛋白偶联受体(GPCRs)及其相关G蛋白的膜结合的空间组织(w/Prosser Lab，Chemical，U of T)。这些项目包括在物理和生命科学的边界对大量HQP进行技术方面的培训和指导，他们将从不同的受训者中积极招聘。 虽然目前仅限于研究实验室，但随着这项技术的成熟，它可能会对工业和生物技术应用产生重大影响，如胰岛素生产、药物筛选和医疗诊断。然而，整个项目在很大程度上依赖于能够快速探测单光子水平光的科学相机。我们目前的EMCCD相机已有10年的历史，使用寿命即将结束，与现代最先进的sCMOS相机相比，速度和灵敏度都要低得多。这严重影响了我们在这个竞争激烈的领域培训HQP和在国际水平上竞争的能力，除非设备升级，否则最终将在未来几年停止这项研究。

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.