A Feasibility Study of a High-Throughput Live-Cell Microscopy Design for Visualizing Viral Particle Action and Nano-Carrier Delivery Performance

用于可视化病毒颗粒作用和纳米载体传递性能的高通量活细胞显微镜设计的可行性研究

• 批准号: 10193517
• 负责人: Haw Yang
• 金额: $ 36.34万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10193517
• 关键词: 3-Dimensional; Address; Alpha Particles; Area; Basic Science; Behavior; Benchmarking; Biochemical; Biological; Capsid; Cell surface; Cells; Communities; Computational algorithm; Computer Assisted; Computer software; Computers; Coupling; Data; Data Set; Detection; Development; Diffuse; Drug Delivery Systems; Endosomes; Event; Exhibits; Feasibility Studies; Genetic Materials; Goals; Human; Image; Individual; Invaded; Knowledge; Laboratories; Laser Scanning Microscopy; Lasers; Location; Machine Learning; Measures; Methods; Microscope; Microscopy; Microtubules; Monitor; Motion; Mutation; Optics; Performance; Research Personnel; Resolution; Sampling; Scanning; Shapes; Supervision; Testing; Therapeutic; Three-Dimensional Imaging; Time; Training; Translational Research; Viral; Virus; Virus-like particle; Visualization; base; data acquisition; data streams; design; experimental study; feasibility testing; imaging modality; imaging platform; insight; instrumentation; interest; live cell microscopy; nanocarrier; nanoparticle; neural network; optical imaging; particle; practical application; progenitor; prototype; spatiotemporal; statistics; tool; trait; two-photon; virology

Project Summary/Abstract Decades of basic research have afforded a general picture for how a viral particle may approach a cell, be internalized by the cell, and hijack the cell to produce more progenitors. The accumulated knowledge, in turn, has allowed one to formulate specific mechanistic questions. For instance, why would a particular mutation in a virus make it more effective in infecting a live cell? Is it because the mutation makes the virus stay on the cell surface longer (on time) but exhibiting the same cell-internalization propensity, or is it because the mutation makes it easier for the virus to invade the cell while the off time remains the same? Where inside a cell and when does a viral particle escape an endosomal enclosure and/or shed its capsid to release its genetic material? By analogy, similar questions can be formulated in designing nanoparticle-based drug delivery vessels. Many ingenious experiments using a diverse array of biochemical and biological tools have been devised to address them, and the insights have led to translational research that has direct therapeutic impacts. A direct observation and recording of these dynamical events that a viral particle may exhibit in its cell-invasion and multiplication cycle could potentially offer much more. Recently, our laboratory put forward a proof-of-principle imaging platform that allows one to do just that: While an integrated two-photon laser-scanning microscope continuously provides 3D sections of the environmental context of a moving virus-like nanoparticle, the nanoparticle is tracked in 3D. This is performed by moving the sample in order to keep the particle at the center of a microscope objective focus with a super-resolution localization precision (~10 nm) in all three dimensions and at a 10-microsecond time resolution. Even for events as simple as a virus-like nanoparticle approaching and landing on a cell, this prototype multiresolution microscope has allowed us to uncover that, unexpectedly, a virus-like particle tends to slow down significantly before its landing on a cell surface. While the prototype instrumentation demonstrates that direct 3D high-resolution visualization could indeed provide uniquely new information that has been inaccessible using conventional methods, its trajectory throughput is too low to be of widespread practical use. This developmental project is intended to test whether a new microscopy design would make this approach higher throughput. This is to be achieved by a new instrumentation design and a machine learning computational backend for dynamic content filtering. Quantitative measures for benchmarking and feasibility testing are also described. If feasible, the community would have a completely new and practical way of looking at viral particle actions and nano-carrier delivery performances.

项目总结/摘要 几十年的基础研究已经为病毒颗粒如何接近细胞提供了一个总体图景， 被细胞内化，并劫持细胞产生更多的祖细胞。积累的知识，反过来， 让我们可以提出具体的机械问题例如，为什么一个特定的突变在一个 使其更有效地感染活细胞？是因为变异使病毒停留在细胞上吗 表面更长（时间），但表现出相同的细胞内化倾向，或者是因为突变 使病毒更容易侵入细胞而关闭时间保持不变？在细胞内的位置和时间 病毒颗粒是否逃离内体外壳和/或脱落其衣壳以释放其遗传物质？通过 类似地，类似的问题可以在设计基于纳米颗粒的药物递送容器中被公式化。许多 已经设计出使用各种生物化学和生物学工具的巧妙实验， 他们，和见解导致了转化研究，有直接的治疗影响。直接观察 并记录病毒颗粒在其细胞侵入和增殖中可能表现出的这些动态事件 循环可以提供更多。最近，我们的实验室提出了一个原理验证成像平台 这使得人们可以做到这一点：而集成的双光子激光扫描显微镜不断提供 移动的病毒样纳米颗粒的环境背景的3D部分，纳米颗粒在3D中被跟踪。 这是通过移动样品以保持颗粒在显微镜物镜的中心来执行的 在所有三个维度上以10微秒的超分辨率定位精度（~10 nm）聚焦 时间分辨率即使是像病毒样纳米粒子接近并降落在细胞上这样简单的事件， 原型多分辨率显微镜使我们能够发现，出乎意料的是，病毒样颗粒往往 在到达细胞表面之前会显著减慢。虽然原型仪器展示了 直接3D高分辨率可视化确实可以提供独特的新信息， 使用常规方法无法实现，其轨迹吞吐量太低而不能广泛实际使用。 这个发展项目的目的是测试是否一个新的显微镜设计将使这种方法 更高的吞吐量。这将通过新的仪器设计和机器学习计算来实现。 用于动态内容过滤的后端。基准测试和可行性测试的量化措施也 介绍了如果可行的话，社会将有一个全新的和实际的方式来看待病毒颗粒 作用和纳米载体递送性能。