Spatiotemporal Corona virus binding dynamics and infection mechanism investigated with 100 Hz ROCS microscopy and thermal fluctuation analysis

冠状病毒感染包括几个连续的步骤，其中扩散接触和结合是病毒进入细胞前的第一个决定性过程。然而，由于只有0.1微米的小病毒颗粒具有极高的动力学，目前的测量技术不可能对进入前的第一个相互作用步骤进行观察和更详细的调查。由于细胞表面粗糙，丝状足常参与病毒的摄取，宿主与病毒的相互作用可能变得更加复杂。本研究计划的目标是揭示病毒的高度动态，不连续的结合过程，这是由位置和方向的热波动所控制的。这个过程可能需要长达30分钟，直到病毒进入宿主。通过使用我们实验室最近开发的基于蓝色激光旋转相干散射（ROCS）的新型无标记超分辨率显微镜方法，我们计划以100 Hz帧速率监测超过100,000张图像的决定性病毒结合过程，而不会损失图像质量。通过自动跟踪和分析病毒在几秒和几分钟内的热位置波动，我们将计算出每个细胞约10-100个病毒结合强度的时间变化。利用这些新的测量和分析方法，我们希望量化伪病毒（带有SARS-COV-19刺突）特异性结合ACE-2受体的开启和关闭结合率——从第一次接触到被吸收到细胞中。通过记录大量的病毒波动轨迹，可以分析具有高统计置信度的接触结合行为。特别是，我们将研究其与表达ACE-2受体的肺上皮细胞和肺巨噬细胞的时空结合行为。在最后一步，我们将测试诸如ACE-2受体阻滞剂等药物对病毒动力学的影响，目的是进一步了解可能的干预机制。

Corona virus infection consists of several successive steps, among which the diffusion contact and binding are the first decisive processes before the virus entries the cell. However due to the extremely high dynamics of the only 0.1 micrometre small virus particles, observations and more detailed investigations of the first interaction steps before entry has not been possible with current measurement technology. The host virus interaction can become even more complicated because of rough cells surfaces, where filopodia are often involved in virus uptake.The goal of this research proposal is to unravel the highly dynamic, discontinuous binding process of viruses, which are governed by thermal fluctuations in position and orientation. This process can take up to 30 min until the virus enters the host. By using the novel label-free, super-resolution microscopy methods based on rotating coherent scattering (ROCS) of blue laser light, which was recently developed in our lab, we plan to monitor the decisive virus binding processes over 100,000 images at 100 Hz frame rate without loss in image quality. By automatic tracking and analysing the thermal position fluctuations of the viruses over seconds and minutes, we will calculate the temporal changes in binding strength of about 10-100 viruses per cell. With these novel measurement and analysis methods we want to quantify the on- and off- binding rate of pseudo-viruses (with SARS-COV-19 spikes) binding specifically to ACE-2 receptors - from the first contact to the uptake into the cell. By recording a large number of virus fluctuation trajectories will allow to analyze the contact binding behavior with high statistic confidence. In particular, we will investigate the spatiotemporal binding behavior to lung epithelial cells und lung macrophages expressing ACE-2 receptors. In a last step, we will test the influence of drugs such as ACE-2 receptor blockers on the virus dynamics with the goal to get further insights in posssible intervention mechanisms.