Luminescent metal complex probes for correlative microscopy

 描述(由申请人提供)：将开发用于相关光学和电子显微镜(CLEM)的新型过渡金属络合物/簇基探针和用于标记整个组织支架的普遍采用的健壮协议。新的探针和方案将使使用一步标记程序标记的两个或更多抗原能够同时定位。在荧光成像之后，通过催化沉积电子致密的银，与光聚合的3，3‘-二氨基联苯胺(DAB)相比，荧光标记的靶标将在电子显微镜中被“看到”，与光聚合的3，3’-二氨基联苯胺(DAB)相比，电子致密的银提供更高的对比度和清晰的点状信号，DAB产生融合信号，需要四氧化三铯(OsO4)。建议的探测器具有以下优点：i)比金探针有更高的量子产率(QY)，与半导体“量子点”(QD)相当的QY；ii)更小的流体动力学半径，毒性更小，在生物缓冲液中比QD更稳定；iii)用于时间分辨成像的长半衰期和大的斯托克斯位移；iv)与使用DAB/OsO4的可光转换荧光蛋白和ReAsH试剂相比，在EM中点状信号和更好的信噪比；v)与一些“超分辨率”技术一起进行成像；以及vi)与遗传编码的可光转换和EM标签一起使用时的相关多路复用。在第一阶段，所提出的探针将用于：1)准确地定位“混合突触”(连接的电和化学突触成分)上的缝隙连接；2)帮助明确地识别在混合突触中发现的一个或两个组成突触蛋白，并用相关的光学和电子显微镜(CLEM)来确定膜的“侧向”。混合突触和特定突触蛋白的定位是有问题的，因为细胞膜及其组成蛋白低于光镜成像技术的分辨率极限。Clem将与波多黎各大学里约热内卢分校生物成像集团的Eduardo Rosa-Molina博士合作进行。罗莎-莫利纳博士一直致力于阐明控制成年男性甘布西亚人极快(20-50毫秒)性交行为的脊髓马达回路，甘布西亚回路是使用新探头进行CLEM的理想测试系统。从长远来看，我们计划开发相关的“超分辨率”显微镜、连续切片电子断层扫描(连续块面/双束扫描电子显微镜)和倾斜系列电子显微镜的试剂和方案。

 DESCRIPTION (provided by applicant): Novel transition metal complex/cluster based probes for correlative light and electron microscopy (CLEM) and universally adoptable robust protocols for labeling whole tissue mounts will be developed. The new probes and protocols will enable simultaneous localization of two or more antigens labeled using a single- step labeling procedure. Following fluorescence imaging, the fluorescently labeled targets with analogs of ruthenium(II)-poly(pyridine) will be made "visible" in the electron microscope by catalyzed deposition of electron dense silver that provides higher contrast and well defined punctuate signal as compared to the photo polymerized 3,3'-diaminobenzidine (DAB), that results in defuse signal and requires osmium tetroxide (OsO4). The proposed probes have following advantages: i) higher quantum yields (QY) than gold probes and comparable QYs to the semiconductor "quantum dots" (QDs); ii) smaller hydrodynamic radii, less toxicity and better stability in biological buffers than QDs; iii) long half-lives and large Stokes shifts for time-resolved imaging; iv) punctate signal and better signal-to-noise ratios in the EM following silver deposition as compared to photoconvertible fluorescent proteins and ReAsH reagents that use DAB/OsO4; v) possible imaging with some of the "super-resolution" techniques; and vi) correlative multiplexing when used with genetically encoded photo-convertible and EM tags. In Phase I, the proposed probes will be used to: 1) accurately locate gap junctions at "mixed synapses" (conjoined electrical and chemical synaptic components) and 2) facilitate the unambiguous identification of one or two constituent synaptic proteins found at mixed synapses and determination of the membrane "sidedness" with correlative light and electron microscopy (CLEM). Localization of mixed synapses and specific synaptic proteins is problematic because cell membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. CLEM will be carried out in collaboration with Dr. Eduardo Rosa- Molinar, Biological Imaging Group, University of Puerto Rico-Rio Piedras. Dr. Rosa-Molinar has been working on elucidating the spinal motor circuitry controlling the adult male Gambusia's extremely rapid (20-50 ms) coital behavior Gambusia's circuitry which is an ideal test system for performing CLEM with the new probes. In the longer term, we plan to develop reagents and protocols for correlative "super-resolution" microscopy, and serial section electron tomography (Serial Block-Face/dual beam SEM) and tilt-series TEM.