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

项目摘要/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大致的图景 被细胞内化，并劫持细胞以产生更多的祖细胞。积累的知识反过来， 使人们能够提出具体的机械性问题。例如，为什么一个特定的突变在一个 病毒使其更有效地感染活细胞？是因为变异使病毒停留在细胞内吗？ 表面更长(准时)，但表现出相同的细胞内化倾向，或者是因为突变 使病毒在关闭时间保持不变的情况下更容易入侵细胞？在牢房内的什么地方以及什么时候 病毒颗粒会逃脱内体包裹体和/或脱下衣壳释放其遗传物质吗？通过 以此类推，在设计基于纳米颗粒的药物输送容器时也可以提出类似的问题。许多 使用各种生化和生物工具的巧妙实验已经被设计出来，以解决 这些见解导致了具有直接治疗影响的转译研究。直观的观察 以及记录病毒颗粒在其细胞入侵和增殖过程中可能表现出的这些动态事件 周期可能会提供更多的东西。近日，我们实验室提出了一种原理验证的成像平台 这使得人们能够做到这一点：当集成的双光子激光扫描显微镜不断提供 移动的类似病毒的纳米颗粒的3D环境部分，该纳米颗粒在3D中被跟踪。 这是通过移动样品来实现的，以便将颗粒保持在显微镜物镜的中心 在所有三个维度的超分辨率定位精度(~10 nm)和10微秒的对焦 时间分辨率。即使是像病毒一样的纳米颗粒接近并降落在细胞上这样简单的事件，这 原型多分辨率显微镜使我们能够发现，出人意料的是，类似病毒的颗粒往往 在它落在细胞表面之前，它会显著减速。虽然原型工具演示了 这种直接的3D高分辨率可视化确实可以提供独特的新信息， 用常规方法无法到达，其轨道吞吐量太低，不能广泛应用。 这一开发项目旨在测试一种新的显微镜设计是否会使这种方法 更高的吞吐量。这将通过一种新的仪器设计和机器学习计算来实现 动态内容过滤的后端。基准和可行性测试的量化措施也是 描述。如果可行，社区将有一种全新而实用的方式来看待病毒颗粒。 行为和纳米载体的输送性能。

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.