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 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现在允许蛮力 定位数千个标记体内特定靶标的单个探针的方法。最基本的 这种成像技术的概念是，单分子可以精确地定位在尺度上 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]。近期 明亮的可光激活、光可切换和自然间歇的荧光探针的发展 允许高密度的单个荧光分子单独成像和精确定位 接近10 nm，导致超分辨率图像，在固定或整齐的静态结构中。这个概念是 如图56所示。SML-SR已经被几个小组[26,298,299]证实，其变异基于 关于所使用的探头、成像条件和分析方法。 基于单分子定位的超分辨技术 (SML-SR)技术结合SPT和单分子 将映像作为最强大的可用工具访问 蛋白质-蛋白质在10 nm尺度上的动态变化 固定单元格。SML-SR的一个重要优势是 其他SR方法(如STED[300]和饱和 图案化激发[301]是标记的位置 可以使用目标(用于生成SR映像) 直接用于分析集群和共聚类AS 在EM中用免疫金标记法完成[11]。 SR-CORE的主要目标是(1)提供以下功能 最先进的单分子荧光技术 STMC和(2)开发新技术，允许 获取更小的空间和时间尺度。这个 高级核心人员将与生物学家密切合作 和分析核心来设计和执行单个 直接回答问题的分子实验 在本提案的其他地方进行了描述。

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.