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.

项目摘要/摘要从分子到电路的大脑结构缺乏全面的地图，这是一个关键的障碍神经科学的进展以及更好，更多的常规方法，以精确定位分子在需要亚细胞水平。脑组织对分子映射提出了双重挑战：除了对高分辨率成像的明显需求，分子的准确定位也需要一种手段可视化周围的细胞和组织结构，不仅识别哪个亚细胞隔室包含一个给定的分子，但哪个细胞。超分辨率荧光显微镜已达到单次分子分辨率，但仅显示探针，而不是组织结构。电子显微镜（EM）很容易揭示亚纳米分辨率下的综合组织结构。 EM水平分子成像的方法，但是，保持效率低下，通常是不可靠的。新开发的转基因方法可以促进 EM对特定目标的定位，但是这些需要遗传操作，提供非常有限的多路复用，并且不揭示内源分子。后置免疫EM，其中抗体标记为直接在EM部分执行，是一种更有效，更通用的方法，但从技术上讲是具有挑战性的是，神经生物学在很大程度上避免了它。邮政的关键独特功能与常规的固定组织免疫标记的方法相比，EM标记是使用用于抗体检测的金颗粒。该提议的前提是黄金探针是一个不足的基于EM部分可调的观察结果，导致后置标记的原因使用简单的常规程序用荧光抗体探针进行标记。与流行荧光相反抗体探针，黄金探针患有不利的化学计量，降低性障碍和不稳定性金抗体复合物。该项目的核心目的是开发用于EM的抗体检测的试剂绕过这些问题的部分。量子点，是半导体纳米晶体 EM可见，是黄金的绝佳替代品，因为它们易于以各种尺寸，形状，和元素组成，既有助于探针优化和多重标记。避免依赖庞大，不稳定的蛋白质金属复合物，限制了灵敏度和信号扩增，A 将使用催化的记者沉积（卡）方法。卡采用抗体连接的过氧化物酶酶以催化探针分子在抗体结合位点上的共价附着。用作卡底物的量子点功能化量子点未取消检测的抗体结合步骤，因此，相对庞大的EM探针不会干扰灵敏度，并且基于酶的探针沉积允许扩增在时间上进行，而不会限制结合位点饱和。这种方法是创新性不仅仅是将一个标签替换为另一个标签，而是解决多个已知的标签现有探针的性能不佳。