UNM SUPER-RESOLUTION CORE

在过去的两年里，荧光显微镜经历了一个数量级的发展， 通过基于单分子的成像技术提高分辨率[296]。技术的演变 探测器、计算能力、分子生物学和荧光探针的发展， 这种方法定位了数千个标记体内特定靶点的单个探针。基本 这种成像技术的概念是，单分子可以以缩放的精度定位 大致为sigma（loc）=sigma psf/N[1/2]，其中sigma（ioc）是定位精度sigma psf是显微镜点扩散的宽度 函数，N是从单个分子收集的光子的预期数量[297]。最近 在明亮的光可激活的、光可切换的和自然间歇的荧光探针方面的发展已经 允许高密度的单个荧光分子精确地单独成像和定位 接近10 nm，导致在固定或接近静态结构中的超分辨率图像。这个概念 如图56所示。SML-SR已被几个小组[26，298，299]证明，其变化基于 对所用探针、成像条件和分析方法的影响。 基于单分子定位的超分辨 （SML-SR）技术将SPT和单分子 成像作为最强大的可用工具， 蛋白质-蛋白质动力学在10 nm尺度的活， 固定细胞SML-SR的一个重要优势是， 其他SR方法（如STED [300]和饱和 [301]第301章：你是谁？ 可以使用目标（用于生成SR图像 直接用于聚类和共聚类分析， 在EM中进行免疫金标记[11]。 SR核心的主要目标是（1）提供这些 现有技术的单分子荧光技术 STMC和（2）开发新技术， 更小的空间和时间尺度。的 SR核心人员将与生物学家密切合作 和分析的核心，设计和执行单 直接回答问题的分子实验 在本提案的其他地方描述。

Over the last two years, fluorescence microscopy has experienced an order of magnitude improvement in resolution via single molecule based imaging techniques [296]. Evolution in the technologies of detectors, computational power, molecular biology and fluorescence probes now allows the brute force approach of localizing thousands of individual probes that are labeling specific targets in vivo. The basic concept of this imaging technique is that single molecules can be localized with an accuracy that scales roughly as sigma(loc)=sigma psf/N[1/2], where sigma (ioc) is the localization accuracy sigma psf is the width of the microscope point spread function, and N is the expected number of collected photons from the single molecule [297]. Recent developments in bright photo-activatable, photo-switchable and naturally intermittent fluorescent probes have allowed high densities of single fluorescent molecules to be imaged and localized individually with accuracy approaching 10 nm, leading to super-resolution images, in fixed or neariy static structures. This concept is illustrated in Figure 56. SML-SR has been demonstrated by several groups [26,298,299] with variations based on the probes used, imaging conditions and analysis approaches. Single Molecule Localization based Super-Resolution (SML-SR) techniques join SPT and single molecule imaging as the most powerful available tools to access protein-protein dynamics at the 10 nm scale in live and fixed cells. One important advantage of SML-SR over other SR approaches (such as STED [300], and Saturated Patterned Excitation [301] is that positions of labeled targets (used to generate the SR images) can be used directly for analysis of clustering and co-clustering as done with immuno-gold labeling in EM [11]. The primary goals of the SR-core are to (1) provide these state of the art single molecule fluorescence techniques to the STMC and (2) develop new techniques that allow access to ever smaller spatial and temporal scales. The SR-core personnel will work closely with the biologists and the analysis core to design and execute single molecule experiments to directly answer questions described elsewhere in this proposal.