Development of green fluorescent protein technology

光激活定位显微镜（PALM）等超分辨率技术使荧光蛋白嵌合体的成像能够在纳米尺度上揭示遗传表达蛋白的组织，其分子密度足够高，可以提供结构背景。在PALM中，对许多光激活荧光蛋白分子的稀疏子集进行连续光激活和随后的漂白。然后，通过点扩散函数的统计拟合来确定单个分子的荧光发射中心，从而在近分子分辨率下定位。来自所有子集的聚合位置信息然后组装成超分辨率图像，其中单个荧光分子在高分子密度（高达10,000个分子/微米平方）下被隔离。我们之前已经在冷冻制备的薄片中展示了细胞内结构（包括溶酶体、高尔基体和线粒体）的PALM成像，以及在TIRF激发下固定细胞中血管蛋白和肌动蛋白的成像，以及线粒体标记蛋白的相关PALM/透射电镜。

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.