Development of green fluorescent protein technology

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纳米级上基因表达的蛋白质的组织结构，分子密度足够高，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。我们之前已经展示了冷冻切片中细胞内结构(包括溶酶体、高尔基体和线粒体)的Palm成像，以及TIRF激发下固定细胞中vinculin和肌动蛋白的成像，以及相关的线粒体标记蛋白的Palm/透射电子显微镜。 我们开发了一种双标记Palm检测系统，该系统使用细胞内表达的两种不同的可光激活分子。此外，我们还开发了一种使用Palm在活细胞中进行单颗粒跟踪的系统，该系统允许在单分子水平上表征蛋白质的扩散和固定。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光激活的荧光蛋白嵌合体的许多子集。通过这种技术成像膜蛋白，可以获得单分子运动的空间分辨地图，提供比传统的单粒子跟踪多几个数量级的每个细胞的轨迹。通过探索不同的分子亚群，包括GAG和VSVG，我们证明了sptPALM可以为探索膜的空间和时间异质性的起源提供一个强大的手段。 我们已经帮助将一种新的干涉超分辨率成像技术应用于生物样品，该技术将单光子多相干涉方案与Palm相结合。这种方法被称为干涉光激活定位显微镜(IPALM)。具体地说，从像可光激活的GFP这样的光子发射器获得的单个光子，在根据不同的路径长度传播后，被允许在三向分束器中自干涉。然后，从分束器输出的三束光束被用来确定源分子的轴向位置，其x-y位置是通过Palm确定的。与散焦技术相比，iPALM的轴向分辨率提高了10倍，光子效率提高了100倍。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沿z轴分辨到几乎它们已知的25纳米尺寸。此外，可以区分质膜前缘的背侧和腹侧位置(50 nm距离)，以及整合素局灶性粘连内的三维结构。IPALM的亚20 nm、3D空间分辨率能力在定量测量蛋白质分布和拓扑结构方面具有很大潜力，这些拓扑结构是细胞内发现的复杂的分子尺度结构的基础。

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. 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.