Development of green fluorescent protein technology

点定位超分辨技术，如光活化定位显微镜(Palm)，使荧光蛋白嵌合体的成像能够揭示基因表达的蛋白质在纳米尺度上的组织结构，其分子密度足以提供结构背景。在Palm中，对大量稀疏的光活化荧光蛋白分子进行连续的光活化和随后的漂白。然后，单个分子通过其点扩散函数的统计拟合来确定它们的荧光发射中心，从而以接近分子的分辨率定位。然后，所有子集的聚集位置信息被组合成超分辨率图像，在该图像中，单个荧光分子以高分子密度(高达10,000个分子/微米的平方)被分离。 虽然Palm是一种研究蛋白质组织的强大方法，但Palm数据集的定量和空间分析工具在很大程度上还缺乏。我们开发了一种与Palm(PC-Palm)的配对相关分析方法，使跨越质膜的蛋白质组织的复杂模式能够被分析。该方法使用一种算法从蛋白质簇中区分具有多种外观的单个蛋白质。这使得能够量化不同的空间组织参数，包括蛋白质簇的存在、它们在质膜中的大小、密度和丰度。利用这种方法，我们在COS-7细胞中展示了具有不同膜锚定和脂分配特征的质膜蛋白的明显纳米级组织，并显示了糖基磷脂酰肌醇(GPI)锚定蛋白在不同扰动下的显著变化。我们的结果表明PC-Palm是一种分析蛋白质异质性和功能的有效工具，具有广泛的适用性，适用于其他单分子策略。 我们开发了一种用于超分辨率成像的定位荧光分子的新方法，该方法不需要可光激活或光开关探测器。这项技术被称为漂白/闪烁辅助定位显微镜(BALM)，它依赖于所有常用荧光探针的固有漂白和闪烁行为。通过获取一系列荧光图像来检测单个荧光团。然后，通过从该系列的每个图像中减去后续图像来记录荧光团漂白或闪烁事件。类似地，通过从每一帧减去前一帧来检测眨眼事件。经过图像相减后，识别单个荧光团的荧光发射信号，并通过用理论高斯分布拟合荧光强度分布来确定局部化。我们发现，香油与所有常用的合成荧光染料和基因表达的荧光蛋白都有效。我们进一步证明，香油可以用于多色超分辨率实验，破译一个样本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超分辨率图像获取方法，既可以单独使用，也可以与其他超分辨率方法结合使用。 受体调节的细胞信号通常是通过形成结构不确定的瞬时、异质蛋白质复合体来调节的。为了更好地了解这一过程，我们使用单色和双色Palm研究了完整T细胞质膜上T细胞抗原受体(TCR)下游的复合体。在静息和激活的细胞中，几个与TCR相关的信号分子，包括LAT、ZAP-70和TCRzeta链，被发现存在于纳米级的簇中，这些簇的形成依赖于蛋白质-蛋白质和蛋白质-脂的相互作用。适配器SLP-76定位于这些簇的外围。这种纳米尺度的结构依赖于聚合的肌动蛋白，它的破坏影响了TCR依赖的细胞功能。 我们致力于一种新的定位显微镜分析方法(称为3B分析)，该方法能够使用标准荧光蛋白和氙弧灯照明极大地提高活细胞荧光图像的分辨率。这项技术使用贝叶斯分析来提高分辨率，方法是对整个数据集的分子的闪烁和漂白行为进行建模，这些分子是由许多荧光团产生的，这些荧光团可能在任何给定的时间发光，也可能不发光。这项技术的高分辨率性能揭示了活细胞内足体形成和解离的纳米尺度动力学，分辨率为50 nm，在4-S时间尺度上。

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.