Luminescent metal complex probes for correlative microscopy

用于关联显微镜的发光金属配合物探针

• 批准号: 8906425
• 负责人: VISHWAS N JOSHI
• 金额: $ 25.25万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8906425
• 关键词: Adult; Amidone; Amines; Antibodies; Antigens; Behavior; Binding; Biological; Biological Process; Biosensing Techniques; Blinking; Buffers; Cell membrane; Cells; Cellular biology; Chemicals; Collaborations; Connexins; Data; Dendrimers; Deposition; Detection; Diffuse; Disease; Electron Microscope; Electron Microscopy; Electrons; Face; Fluorescence; Fluorescent Dyes; Gambusias; Gap Junctions; Gold; Gold Colloid; Heavy Metals; Heme; Image; Imagery; Imaging Techniques; Immunoglobulin Fragments; In Vitro; Individual; Ions; Label; Life; Light; Membrane; Methods; Microscopic; Microscopy; Modification; Molecular; Molecular Biology; Motor; Natural regeneration; Noise; Organelles; Osmium Tetroxide; Penetration; Phase; Phenols; Piedra; Preparation; Procedures; Process; Proteins; Protocols documentation; Puerto Rico; Quantum Dots; Radial; Reagent; Reducing Agents; Research Personnel; Resolution; Rosa; Ruthenium; Sampling; Semiconductors; Series; Signal Transduction; Silver; Slide; Specimen; Spinal; Structure; Synapses; System; Techniques; Testing; Thick; Time; Tissues; Toxic effect; Transition Elements; Universities; Work; analog; antibody conjugate; base; electron tomography; fluorescence imaging; improved; in vivo; light microscopy; luminescence; macromolecule; male; metal complex; nanometer; novel; oxidation; public health relevance; pyridine; quantum; sample fixation; structural biology; surface coating

 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.

 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/簇基探针以及用于标记整个组织样品的普遍采用的稳健方案。新的探针和方案将使两个或两个以上的抗原标记使用一个单一的步骤标记程序的同时定位。在荧光成像之后，通过电子致密银的催化沉积，用钌（II）-聚（吡啶）的类似物荧光标记的靶将在电子显微镜中“可见”，与光聚合的3，3 '-二氨基联苯胺（DAB）相比，所述银提供更高的对比度和良好限定的点状信号，所述光聚合的3，3'-二氨基联苯胺（DAB）导致缓和信号并需要四氧化锇（OsO 4）。 所提出的探针具有以下优点：i）比金探针更高的量子产率（QY）和与半导体“量子点”（QD）相当的QY; ii）比QD更小的流体动力学半径、更小的毒性和在生物缓冲液中更好的稳定性; iii）用于时间分辨成像的长半衰期和大的Stokes位移; iv）与使用DAB/OsO 4的光可转换荧光蛋白和ReAsH试剂相比，在银沉积后的EM中具有点状信号和更好的信噪比; vi）当与遗传编码的光可转换和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）准确定位间隙连接在“混合突触”（联合的电和化学突触组件）和2）促进明确的识别一个或两个组成突触蛋白发现在混合突触和膜的“侧性”与相关的光和电子显微镜（CLEM）的确定。混合突触和特定突触蛋白的定位是有问题的，因为细胞膜和它们的组成蛋白低于光学显微镜成像技术的分辨率极限。CLEM将与波多黎各-里奥皮德拉斯大学生物成像组的Eduardo Rosa-Molinar博士合作进行。Rosa-Molinar博士一直致力于阐明控制成年雄性食蚊鱼极快（20-50 ms）性交行为的脊髓运动回路，食蚊鱼的回路是用新探针进行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SEM）和倾斜系列TEM的试剂和协议。