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; 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（由应用提供）：将开发出用于校正光和电子显微镜（CLEM）的新型过渡金属复合物/群集项目，并将开发用于标记整个组织安装座的标签的通用适应性稳健方案。新问题和协议将使使用单步标记过程标记的两个或多个抗原的简单定位。 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 defense signal and requires osmium四氧化物（OSO4）。所提出的探针具有以下优点：i）比金探针更高的量子收益率（QY），而与半导体“量子点”（QDS）相当的Qys； ii）较小的流体动力半径，比QDS较小的毒性和更好的生物缓冲稳定性； iii）长的半衰期和大的stok旋转，以进行时间分辨的成像； iv）与使用DAB/OSO4的可光转换荧光蛋白和REASH试剂相比，在银沉积之后，EM中的点状信号和更好的信噪比； v）可能使用一些“超分辨率”技术进行成像；和vi）相关的多路复用，当与一般编码的光转换和EM标签一起使用时。在第一阶段，提出的问题将用于：1）准确定位“混合突触”（连接的电气和化学突触组件）和2）促进对一个或两个组成的突触蛋白的明确鉴定，该突触蛋白建立在混合的突触上，并确定膜的“层次”和corlational sircipoption（crem clermosc）。混合突触和特定合成蛋白的定位是有问题的，因为细胞膜及其组成蛋白低于光显微成像技术的分辨率。 CLEM将与波多黎各大学皮埃拉斯大学生物成像集团Eduardo Rosamolinar博士合作进行。 Rosa-Molinar博士一直致力于阐明控制成年男性Gambusia极快（20-50毫秒）外套行为Gambusia的电路的脊柱电机电路，这是对具有新问题进行CLEM的理想测试系统。从长远来看，我们计划开发相关“超分辨率”显微镜的试剂和方案，以及串行部分电子断层扫描（串行块/双束光束SEM）和倾斜序列TEM。