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Quantum dot probes for electron microscopy

用于电子显微镜的量子点探针

基本信息

批准号：
10043302
负责人：
LINNAEA E OSTROFF
金额：
$ 44.28万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10043302
关键词：
Address Antibodies Antibody Binding Sites Antigens Architecture Attention Axon Binding Binding Sites Blocking Antibodies Brain Brain Mapping Caliber Cells Cellular Structures Collection Complex Crystallization Dendrites Dendritic Spines Deposition Detection Development Electron Microscopy Enzymes Failure Fluorescence Fluorescence Microscopy Fluorescent Antibody Technique Goals Gold Gold Colloid Image Immunoelectron Microscopy Immunofluorescence Immunologic Individual Label Life Link Literature Maps Methods Molecular Neurobiology Neurons Neurosciences Paraffin Embedding Pathology Performance Periodicity Peroxidases Plant Resins Procedures Process Proteins Protocols documentation Publishing Quantum Dots Reagent Reporter Resolution Sample Size Sampling Semiconductors Shapes Signal Transduction Stains Structure Synapses System Techniques Thinness Time Tissues Transgenic Organisms base brain cell brain tissue design experimental study fluorescence microscope fluorophore genetic manipulation high resolution imaging imaging approach imaging modality improved innovation metal complex molecular imaging nanocrystal nanometer nanometer resolution neglect nervous system disorder particle sample fixation single molecule stoichiometry tool

项目摘要

Project Summary/Abstract The lack of comprehensive maps of brain architecture from molecules to circuits is a critical barrier to progress in neuroscience, and better, more routine methods for accurately localizing molecules at the subcellular level are needed. Brain tissue presents a twofold challenge for molecular mapping: in addition to the obvious need for high-resolution imaging, accurate localization of molecules also requires a means of visualizing the surrounding cellular and tissue structure to identify not only which subcellular compartment contains a given molecule, but which cell. Super-resolution fluorescence microscopy has achieved single- molecule resolution, but reveals only probes, not tissue structure. Electron microscopy (EM) readily reveals comprehensive tissue structure at sub-nanometer resolution. Methods for molecular imaging at the EM level, however, remain inefficient and are often unreliable. Newly developed transgenic approaches can facilitate localization of specific targets by EM, but these require genetic manipulation, offer very limited multiplexing, and do not reveal endogenous molecules. Postembedding immuno-EM, in which antibody labeling is performed directly on EM sections, is a much more efficient and versatile approach, but is technically challenging to the point that it is largely avoided in neurobiology. A crucial unique feature of postembedding EM labeling, in contrast to the routine, widely used methods for immunolabeling of fixed tissue, is the use of gold particles for antibody detection. The premise of this proposal is that gold probes are an underappreciated cause of failure in postembedding labeling, based on the observation that EM sections are amenable to labeling with fluorescent antibody probes using simple, routine procedures. In contrast to popular fluorescent antibody probes, gold probes suffer from unfavorable stoichiometry, stearic hinderance, and instability of the gold-antibody complexes. The central aim of this project is to develop reagents for antibody detection on EM sections that circumvent these problems. Quantum dots, which are semiconductor nanocrystals that are visible by EM, are an excellent alternative to gold as they are simple to synthesize in a variety of sizes, shapes, and elemental compositions, which facilitates both probe optimization and multiplexed labeling. To avoid reliance on bulky, unstable protein-metal complexes that limit both sensitivity and signal amplification, a catalyzed reporter deposition (CARD) approach will be used. CARD employs antibody-linked peroxidase enzymes to catalyze covalent attachment of probe molecules to proteins at the antibody binding site. Functionalizing quantum dots for use as CARD substrates uncouples the antibody binding step from detection, so that the relatively bulky EM probe does not interfere with sensitivity, and enzyme-based probe deposition allows amplification to proceed across time without the limitation of binding-site saturation. This approach is innovative in that it does not simply replace one label for another, but instead addresses multiple known causes of poor performance in the existing probes.

项目概要/摘要缺乏从分子到电路的大脑结构的全面图谱是实现这一目标的一个关键障碍。神经科学的进展，以及更好、更常规的方法来准确定位分子需要亚细胞水平。脑组织对分子作图提出了双重挑战：除了显然需要高分辨率成像，分子的精确定位还需要一种手段可视化周围的细胞和组织结构，不仅可以识别哪个亚细胞区室包含给定的分子，但包含哪个细胞。超分辨率荧光显微镜已实现单分子分辨率，但仅显示探针，而不显示组织结构。电子显微镜（EM）很容易揭示亚纳米分辨率的综合组织结构。电磁水平分子成像方法，然而，它们仍然效率低下并且往往不可靠。新开发的转基因方法可以促进通过 EM 定位特定目标，但这些需要基因操作，提供的多重性非常有限，并且不揭示内源性分子。包埋后免疫电镜，其中抗体标记直接在 EM 切片上执行，是一种更高效、更通用的方法，但在技术上具有挑战性，以至于在神经生物学中基本上避免了这种情况。后嵌入的一个重要的独特特征与常规、广泛使用的固定组织免疫标记方法相比，EM 标记是使用用于抗体检测的金颗粒。该提案的前提是黄金勘探是一个被低估的项目嵌入后标记失败的原因，基于 EM 切片适合的观察使用简单的常规程序用荧光抗体探针进行标记。与流行的荧光灯相比抗体探针、金探针都存在不利的化学计量、空间位阻和不稳定的问题金-抗体复合物。该项目的中心目标是开发用于 EM 抗体检测的试剂规避这些问题的部分。量子点是一种半导体纳米晶体通过 EM 可见，它们是黄金的绝佳替代品，因为它们易于合成各种尺寸、形状、和元素组成，这有利于探针优化和多重标记。为了避免对庞大且不稳定的蛋白质-金属复合物的依赖限制了灵敏度和信号放大，将使用催化报告沉积（CARD）方法。 CARD 采用抗体连接的过氧化物酶酶催化探针分子与抗体结合位点的蛋白质共价连接。将量子点功能化用作 CARD 基质，将抗体结合步骤与检测分离，这样相对庞大的 EM 探针就不会干扰灵敏度和基于酶的探针沉积允许扩增跨时间进行，而不受结合位点饱和度的限制。这种方法是创新之处在于它不是简单地将一个标签替换为另一个标签，而是解决了多个已知的问题现有探头性能不佳的原因。