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.