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载片的普遍采用的稳健协议。新的探针和方案将能够使用单步标记程序同时定位两种或多种标记的抗原。荧光成像后，通过电子致密银的催化沉积，带有钌 (II)-聚吡啶类似物的荧光标记靶标将在电子显微镜中“可见”，与光聚合 3,3'-二氨基联苯胺 (DAB) 相比，该银可提供更高的对比度和明确的点状信号，从而产生消散信号并需要锇 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产率（QY）以及与半导体“量子点”（QD）相当的QY； ii) 与量子点相比，流体动力学半径更小，毒性更小，在生物缓冲液中的稳定性更好； iii) 时间分辨成像的长半衰期和大斯托克斯位移； iv) 与使用 DAB/OsO4 的光转换荧光蛋白和 ReAsH 试剂相比，银沉积后的 EM 中具有点状信号和更好的信噪比； v) 使用某些“超分辨率”技术进行可能的成像； vi) 与基因编码的光转换和 EM 标签一起使用时的相关复用。 在第一阶段，所提出的探针将用于：1）准确定位“混合突触”（连接的电和化学突触成分）处的间隙连接，2）促进明确识别混合突触中发现的一种或两种构成突触蛋白，并通过相关光学和电子显微镜（CLEM）确定膜“侧面”。混合突触和特定突触蛋白的定位存在问题，因为细胞膜及其组成蛋白低于光学显微成像技术的分辨率极限。 CLEM 将与波多黎各里奥彼德拉斯大学生物成像小组的 Eduardo Rosa- Molinar 博士合作开展。 Rosa-Molinar 博士一直致力于阐明控制成年雄性 Gambusia 极快（20-50 毫秒）性交行为的脊髓运动电路。Gambusia 的电路是使用新探针执行 CLEM 的理想测试系统。从长远来看，我们计划开发用于相关“超分辨率”显微镜、串行切片电子断层扫描（串行块面/双束 SEM）和倾斜系列 TEM 的试剂和方案。