Development of green fluorescent protein technology

点定位超分辨技术，如光激活定位显微镜（PALM），使荧光蛋白嵌合体的成像，以揭示在纳米级的分子密度高到足以提供结构背景的遗传表达的蛋白质的组织。在PALM中，进行光活化荧光蛋白分子的许多稀疏子集的连续光活化和随后的漂白。然后，通过对单个分子的点扩散函数进行统计拟合来确定其荧光发射中心，从而以接近分子的分辨率定位单个分子。然后，来自所有子集的聚集位置信息被组装成超分辨率图像，其中单个荧光分子以高分子密度（高达10，000个分子/平方微米）被分离。 虽然PALM是研究蛋白质组织的强大方法，但PALM数据集的定量空间分析工具在很大程度上缺失。我们开发了一种与PALM（PC-PALM）的配对相关分析方法，该方法能够分析跨质膜的蛋白质组织的复杂模式。该方法使用一种算法来区分具有多种外观的单个蛋白质和蛋白质簇。这使得能够量化空间组织的不同参数，包括蛋白质簇的存在、它们在质膜中的大小、密度和丰度。使用这种方法，我们证明了不同的膜锚定和脂质分配特性在COS-7细胞的质膜蛋白质的不同的纳米级组织，并表现出显着的变化，糖基磷脂酰肌醇（GPI）锚定的蛋白质的安排在不同的扰动。我们的研究结果表明，PC-PALM是一个有效的工具，具有广泛的适用性，分析蛋白质的异质性和功能，适用于其他单分子策略。 我们开发了一种定位荧光分子的新方法，用于超分辨率成像，不需要光活化或光开关探针。称为漂白/闪烁辅助定位显微镜（BaLM），该技术依赖于所有常用荧光探针的固有漂白和闪烁行为特征。通过获取荧光图像流来检测单个荧光团。然后通过从该系列的每个图像中减去随后的图像来记录荧光团漂白或熄灭事件。类似地，通过从每个帧中减去前一帧来检测闪烁开启事件。在图像相减之后，识别来自单个荧光团的荧光发射信号，并且通过用理论高斯拟合荧光强度分布来确定定位。我们发现BaLM与所有常用的合成荧光染料和遗传表达的荧光蛋白一起工作。我们进一步表明，BaLM可用于超分辨实验，破译样品中多达四种不同蛋白质的分子分布。这些特点表明，BaLM是一种实用和通用的方法，用于获得超分辨率图像，可以单独使用或与其他超分辨率方法结合使用。 受体调节的细胞信号传导通常由结构不确定的瞬时异质蛋白质复合物的形成介导。为了更深入地了解这一过程，我们使用单色和双色PALM来研究完整T细胞质膜上T细胞抗原受体（TCR）下游的复合物。在静息和活化的细胞中，发现几种TCR相关的信号分子，包括LAT、ZAP-70和TCR zeta链存在于纳米级簇中，其形成依赖于蛋白质-蛋白质和蛋白质-脂质相互作用。适配器SLP-76定位于这些簇的外围。这种纳米级结构依赖于聚合的肌动蛋白，其破坏影响TCR依赖性细胞功能。 我们促成了一种新的本地化显微镜分析方法（称为3B分析），能够大大提高使用标准荧光蛋白和氙弧灯照明的活细胞荧光图像的分辨率。该技术采用贝叶斯分析来提高分辨率，方法是对来自整个数据集的分子的闪烁和漂白行为进行建模，该数据集由许多荧光团生成，这些荧光团在任何给定时间可能会发光，也可能不会发光。该技术的高分辨率性能揭示了在活细胞内以50 nm的分辨率在4-s时间尺度上的足状体形成和解离的纳米级动力学。

Point-localization superresolution techniques such as photoactivated localization microscopy (PALM) enable the imaging of fluorescent protein chimeras to reveal the organization of genetically-expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. In PALM, serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules is performed. Individual molecules are then localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The aggregate position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities (up to 10,000 molecules/micron squared). While PALM is a powerful approach for investigating protein organization, tools for quantitative, spatial analysis of PALM datasets are largely missing. We developed a pair-correlation analysis method with PALM (PC-PALM) that enables complex patterns of protein organization across the plasma membrane to be analyzed. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrated distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and showed dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. Our results revealed that PC-PALM is an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies. We developed a new way of localizing fluorescent molecules for superresolution imaging that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. Single fluorophores are detected by acquiring a stream of fluorescence images. Fluorophore bleach or blink-off events are then recorded by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We found that BaLM works with all commonly used synthetic fluorescent dyes and genetically expressed fluorescent proteins. We further showed that BaLM can be used in multicolor superresolution experiments, deciphering the molecular distribution of up to four different proteins in a sample. These characteristics indicated that BaLM is a practical and versatile approach for obtaining superresolution images that can either stand alone or be used in conjunction with other superresolution approaches. Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. To obtain greater insight into this process, we used single and two-color PALM to study complexes downstream of the T cell antigen receptor (TCR) at the plasma membrane of intact T cells. In resting and activated cells, several TCR associated signaling molecules, including LAT, ZAP-70 and TCRzeta chain were found to reside in nanoscale clusters whose formation depended on protein-protein and protein-lipid interactions. The adaptor SLP-76 localized to the periphery of these clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. We contributed to a new localization microscopy analysis method (called 3B analysis) that is able to greatly improve the resolution of live cell fluorescent images using standard fluorescent proteins and xenon arc lamp illumination. The technique employs Bayesian analysis to improve resolution by modeling the blinking and bleaching behavior of molecules from an entire dataset being generated by a number of fluorophores that may or may not be emitting light at any given time. The high resolution performance of this technique revealed the nanoscale dynamics of podosome formation and dissociation within a live cell with a resolution of 50 nm on a 4-s timescale.