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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.

项目总结/摘要缺乏从分子到电路的全面的大脑结构图是一个关键的障碍，神经科学的进步，以及更好的，更常规的方法来准确地定位分子，需要亚细胞水平。脑组织对分子作图提出了双重挑战：除了对高分辨率成像的明显需求，分子的精确定位也需要一种手段，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，包含一个给定的分子，但细胞。超分辨率荧光显微镜已经实现了单- 分子分辨率，但只显示探针，而不是组织结构。电子显微镜（EM）很容易揭示亚纳米分辨率的全面组织结构。EM水平的分子成像方法，而且往往不可靠。新开发的转基因方法可以促进通过EM定位特定靶标，但这些需要遗传操作，提供非常有限的多重化，并且不显示内源性分子。着床后免疫-EM，其中抗体标记是直接在EM切片上执行，是一种更有效和通用的方法，但在技术上这一点在神经生物学中基本上是避免的。植入后的一个重要特征 EM标记，与常规的广泛使用的固定组织免疫标记方法相反，是使用用于抗体检测的金颗粒。这项建议的前提是，黄金探头是一个低估植入后标签失败的原因，基于观察结果，即EM切片符合使用简单的常规程序用荧光抗体探针标记。与流行的荧光灯相比，抗体探针、金探针遭受不利的化学计量、硬脂酸阻碍和不稳定性。金抗体复合物本项目的中心目标是开发EM抗体检测试剂规避这些问题的部分。量子点是半导体纳米晶体，通过EM可见，是金的极好替代品，因为它们可以简单地合成为各种尺寸，形状，和元素组成，这有利于探针优化和多重标记。避免依赖于庞大的、不稳定的蛋白质-金属复合物，限制了灵敏度和信号放大，将使用催化的报告物沉积（CARD）方法。CARD使用抗体连接的过氧化物酶酶催化探针分子与蛋白质在抗体结合位点的共价连接。用作CARD基底的官能化量子点使抗体结合步骤与检测分离，从而相对庞大的EM探针不干扰灵敏度，并且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的限制。这种方法其创新之处在于，它不是简单地用一个标签替换另一个标签，而是解决了多个已知的现有探头性能差的原因。