HPF-X: High-pressure freezing with buffer exchange

项目总结摘要 配体触发的事件在神经科学、内分泌学、病毒学、免疫学、 和药理学。然而，刺激之后的分子和超微结构的变化很难 可视化是因为它们涉及到快速的纳米级运动和蛋白质和膜的修饰。现状-- 最先进的技术不足以捕捉这些时空变化。例如，实时荧光 成像受到空间分辨率(衍射限制)和标记限制(无抗体访问或 在活细胞中洗涤)，而纳米成像方法要么缺乏时间分辨率来捕捉快速动力学 (如超分辨率光学显微镜)或与活细胞成像完全不兼容(如标准或 低温电子显微镜；膨胀显微镜)。考虑到这些限制，时间分辨的冷冻玻璃化 这种方法是通过冷冻样本在刺激后确定的等待时间后捕捉细胞过程的理想方法 在成像之前处于无定形冰的状态。高压冷冻(HPF)通常用于这一目的 由于其样品厚度限制较宽松(&lt；300微米)，与大气中的低温骤降相比 压力(&lt；10微米)。但是，与时间分辨缓冲区交换兼容的HPF设备目前不支持 是存在的。为此，我们将开发一种具有时间分辨缓冲区交换能力的HPF-X设备 在冷冻玻璃化之前。缓冲液交换将允许用各种生物和 包括离子、小分子、多肽和蛋白质(例如，激素、细胞因子， 抗体和纳米抗体)，甚至病毒和细胞。因此，HPF-X将允许冷冻玻璃化细胞、组织 样品，或在用配体刺激后的一系列时间点上的整个小生物体 利用电子显微镜和超分辨率光学等纳米级成像技术进行讯问 显微镜。这种方法将允许在纳米尺度的空间捕捉配体触发的细胞过程 &lt；50毫秒的分辨率和时间分辨率。这项技术的生物学应用包括纳米级成像。 蛋白质-蛋白质相互作用、翻译后修饰和蛋白质-膜动力学。尽管一个 从根本上说，需要新的HPF仪器设计来允许缓冲交换，我们广泛的初步数据 确认了可行性。在目标1中，我们将开发一种与缓冲交换和 冷冻玻璃化，并对其性能进行表征。在目标2中，我们将开发一种时间分辨低温的方法。 使用金标生物样本对系统进行冷却和验证。HPF-X的研制迫在眉睫 纳米生物成像技术的出现带来了技术机遇。重要的是，这项工作超出了当前的范围 冷冻玻璃化领域的方法开发制度，因为所有可用的HPF设备都是商业化的。我们的 定制的HPF-X仪器将允许完全控制、多功能性，并便于其他设备采用和修改 研究人员基于他们的项目需求，这是使用现成的HPF仪器无法实现的。

PROJECT SUMMARY ABSTRACT Ligand-triggered events are central to many processes in neuroscience, endocrinology, virology, immunology, and pharmacology. However, molecular and ultrastructural changes that follow the stimulus are difficult to visualize because they involve rapid nanoscale motions and modifications of proteins and membranes. State-of- the-art techniques are insufficient to capture these spatiotemporal changes. For example, live fluorescence imaging is limited by the spatial resolution (diffraction-limit) and labeling constraints (no antibody access or washing in live cells), while nanoscale imaging methods either lack temporal resolution to capture fast dynamics (e.g., super-resolution optical microscopy) or are incompatible with live-cell imaging altogether (e.g., standard or cryo-electron microscopy; expansion microscopy). Given these limitations, time-resolved cryo-vitrification methods are ideal for capturing cellular processes after a defined wait time post-stimulation by freezing samples in the state of amorphous ice prior to imaging. High-pressure freezing (HPF) is often used for this purpose because of its relaxed sample thickness constraints (<300 µm) as compared to cryo-plunging at atmospheric pressure (<10 µm). However, an HPF device compatible with time-resolved buffer exchange does not currently exist. To this end, we will develop HPF-X – an HPF device with a capability for time-resolved buffer exchange preceding cryo-vitrification. Buffer exchange will allow stimulating the sample with various biological and pharmacological agents including ions, small molecules, peptides and proteins (e.g., hormones, cytokines, antibodies, and nanobodies), and even viruses and cells. Thus, HPF-X will allow cryo-vitrifying cells, tissue samples, or entire small organisms at a series of time points following stimulation with ligands for subsequent interrogation with nanoscale imaging techniques such as electron microscopy and super-resolution optical microscopy. This approach will allow capturing ligand-triggered cellular processes with nanoscale spatial resolution and temporal resolution of <50 ms. Biological applications of this technique include nanoscale imaging of protein-protein interactions, post-translational modifications, and protein-membrane dynamics. Although a fundamentally new HPF instrument design is required to allow buffer exchange, our extensive preliminary data confirms feasibility. In Aim 1, we will develop a high-pressure chamber compatible with buffer exchange and cryo-vitrification and characterize its performance. In Aim 2, we will develop a method for time-resolved cryo- cooling and validate the system using gold-standard biological samples. Development of HPF-X is an emergent technical opportunity given the advent of nanoscale bioimaging. Importantly, this work goes beyond the current method development regime in cryo-vitrification field because all available HPF devices are commercial. Our custom-built HPF-X instrument will allow full control, versatility, and ease of adoption and modification by other researchers based on their project needs, which cannot be achieved with off-the-shelf HPF instruments.