Development of green fluorescent protein technology

超分辨率技术，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。我们以前已经证明了PALM成像的细胞内结构（包括溶酶体，高尔基体和线粒体）在冷冻制备的薄切片，以及在固定的细胞与TIRF激发，和相关的PALM/透射电子显微镜的线粒体标记蛋白质的黏着斑蛋白和肌动蛋白的成像。 我们已经开发了一种双标记PALM检测系统，使用两种不同的细胞内表达的光活化分子。此外，我们已经开发了一个系统，用于在活细胞中使用PALM进行单粒子跟踪，该系统允许在单分子水平上表征蛋白质扩散和固定。这项技术被称为单粒子跟踪PALM（sptPALM），涉及激活、定位和漂白活细胞中许多光活化荧光蛋白嵌合体的子集。单分子运动的空间分辨图可以通过用这种技术对膜蛋白进行成像来获得，比传统的单粒子跟踪提供每个细胞多几个数量级的轨迹。通过探测不同的分子子集，包括Gag和VSVG，我们证明了sptPALM可以为探索膜中时空异质性的起源提供有力的手段。 我们已经帮助应用于生物样品的一种新的干涉超分辨率成像技术，集成了单光子多相干涉计划与PALM。这种方法被称为干涉光激活定位显微镜（iPALM）。具体地，来自光子发射器（如可光活化GFP）的单个光子在行进不同的路径长度之后，允许在3路分束器中自干涉。然后使用来自分束器的三个输出光束来确定源分子的轴向位置，其x-y位置经由PALM确定。与散焦技术相比，iPALM提供了轴向分辨率的10倍改进和光子效率的100倍改进。这使得它特别适合于PA-FP的精确3D定位。iPALM成像已经将微管的直径分辨为几乎它们已知的沿z轴沿着25 nm的尺寸。此外，可以区分质膜前缘处的背侧和腹侧位置（50 nm距离），并且可以区分粘着斑内v整联蛋白的3D组织（Shtengel et al.，2009年）。iPALM的亚20 nm，3D空间分辨率能力对于定量测量细胞内发现的复杂分子尺度结构的蛋白质分布和拓扑结构具有很大的潜力。

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). We have previously demonstrated PALM imaging of intracellular structures (including lysosome, Golgi apparatus and mitochondria) in cryo-prepared thin sections, as well as imaging of vinculin and actin in fixed cells with TIRF excitation, and correlative PALM/transmission electron microscopy of a mitochondrial marker protein. We have developed a dual-label PALM assay system using two different photactivatable molecules expressed within cells. In addition, we have developed a system for doing single particle tracking using PALM in living cells that allows protein diffusion and immobilization to be characterized at the single molecule level. Called single particle tracking PALM (sptPALM), the technique involves activating, localizing and bleaching many subsets of photoactivatated fluorescent protein chimeras in live cells. Spatially-resolved maps of single molecule motions can be obtained by imaging membrane proteins with this technique, providing several orders of magnitude more trajectories per cell than by traditional single particle tracking. By probing distinct subsets of molecules, including Gag and VSVG, we demonstrated that sptPALM can provide a powerful means for exploring the origin of spatial and temporal heterogeneities in membranes. We have helped apply to biological samples a new interferometry superresolution imaging technique that integrates a single-photon multiphase interferometric scheme with PALM. This approach is called interferometric photoactivated localization microscopy (iPALM). Specifically, a single photon derived from a photon emitter like photoactivatable GFP, after traveling different path lengths dependent, is allowed to self-interfere in a 3-way beam splitter. The three output beams from the splitter are then used to determine the axial position of the source molecule, whose x-y position is determined via PALM. iPALM provides a 10-fold improvement in axial resolution and a 100-fold improvement in photon efficiency compared to defocusing techniques. This makes it is particularly suited for accurate 3D localization of PA-FPs. iPALM imaging has resolved the diameter of microtubules to nearly their known dimension of 25-nm along the z-axis. In addition, the dorsal and ventral positions at the leading edge of the plasma membrane (50-nm distance) could be distinguished, and the 3D organization of αv integrin within focal adhesions (Shtengel et al., 2009). The sub-20 nm, 3D spatial resolution capability of iPALM has much potential for quantitative measurements of protein distributions and topologies that underlie the complex, molecular-scale structures found within cells.