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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要数十年的基础研究为病毒粒子如何接近细胞提供了一般图片，由细胞内化，劫持细胞以产生更多的祖细胞。积累的知识反过允许一个人提出特定的机械问题。例如，为什么特定的突变会在病毒使其在感染活细胞方面更有效？是因为突变使病毒留在细胞上表面更长（准时），但表现出相同的细胞内部化倾向，或者是因为突变使病毒更容易在休息时间保持不变时入侵细胞吗？在牢房内以及何时何时病毒颗粒是否逃脱一个内体围栏和/或脱落其capsid以释放其遗传物质？经过类比，在设计基于纳米颗粒的药物输送容器时可以提出类似的问题。许多已经设计了使用各种生化和生物学工具的巧妙实验来解决他们以及见解导致了具有直接治疗影响的转化研究。直接观察并记录病毒颗粒可能在其细胞摄入和繁殖中表现出的这些动力事件周期可能会提供更多。最近，我们的实验室提出了一个原则成像平台这允许一个人这样做：虽然一个集成的两光子激光扫描显微镜不断提供 3D纳米颗粒的环境环境环境环境的3D部分，纳米颗粒以3D跟踪。这是通过移动样品来执行的，以使粒子保持在显微镜物镜的中心在所有三个维度和10英寸美动中的超级分辨率定位精度（〜10 nm）的焦点时间分辨率。即使对于像病毒状的纳米颗粒接近和降落在细胞上的简单事件，原型多分辨率显微镜使我们能够发现，出乎意料的是，病毒样粒子倾向于在降落在细胞表面之前，请大大减速。虽然原型仪器证明了直接的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访问，其轨迹吞吐量太低而无法实用。该开发项目旨在测试新的显微镜设计是否可以使这种方法更高的吞吐量。这是通过新的仪器设计和机器学习计算来实现的动态内容过滤的后端。基准测试和可行性测试的定量措施也是描述。如果可行动作和纳米载体交付性能。