HPF-X: High-pressure freezing with buffer exchange

HPF-X：带有缓冲液交换的高压冷冻

• 批准号: 10704139
• 负责人: Maxim Prigozhin
• 金额: $ 31.89万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10704139
• 关键词: Adoption; Antibodies; Atmospheric Pressure; Biological; Biological Process; Buffers; Cell physiology; Cells; Coupling; Cryo-electron tomography; Cryoelectron Microscopy; Custom; Data; Development; Devices; Diamond; Disease; Electron Microscopy; Endocrinology; Endocytosis; Ethane; Event; Freezing; Future; Goals; Health; Hormones; Ice; Image; Imaging Techniques; Imaging ligands; Immunology; Ions; Label; Lifting; Ligands; Liquid substance; Maps; Membrane; Membrane Proteins; Methods; Microfluidics; Microscopy; Modification; Molecular; Motion; Neurosciences; Optics; Organism; Outcome; Peptides; Performance; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Post-Translational Protein Processing; Process; Proteins; Qualifying; Relaxation; Research Personnel; Resolution; Sample Size; Sampling; Series; Specific qualifier value; Stimulus; System; Techniques; Technology; Thermal Conductivity; Thick; Time; Tissue Sample; Tissues; Validation; Virus; Visualization; Wait Time; Work; bioimaging; biological research; chemical fixation; cryogenics; cytokine; design; fluorescence imaging; high resolution imaging; imaging modality; improved; innovation; instrument; live cell imaging; meter; method development; nanobodies; nanoscale; pharmacologic; preservation; pressure; protein protein interaction; prototype; receptor; response; small molecule; spatiotemporal; superresolution microscopy; temporal measurement; tool; ultra high resolution; virology

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.

项目摘要摘要 配体触发的事件对于神经科学，内分泌学，病毒学，免疫学， 和药理学。但是，刺激遵循的分子和超微结构变化很难 可视化是因为它们涉及快速的纳米级运动以及蛋白质和膜的修饰。最新 艺术技术不足以捕获这些时空变化。例如，实时荧光 成像受到空间分辨率（衍射限制）和标记约束的限制（无抗体访问或 在活细胞中洗涤），而纳米级成像方法要么缺乏临时分辨率来捕获快速动态 （例如，超分辨率光学显微镜）或完全与实时成像不兼容（例如，标准或 冷冻电子显微镜；扩展显微镜）。鉴于这些局限 方法是通过冷冻样品在刺激后定义的等待时间后捕获细胞过程的理想选择 在成像之前处于无定形冰状态。高压冻结（HPF）通常用于此目的 由于其放松的样品厚度限制（<300 µm），与大气处的冷冻策划相比 压力（<10 µm）。但是，HPF设备与时间分辨的缓冲区交换兼容 存在。为此，我们将开发HPF-X - 具有时间分辨缓冲区交换功能的HPF设备 在冷冻剂量之前。缓冲区交换将允许使用各种生物学和 包括离子，小分子，肽和蛋白质在内的药理学剂（例如激素，细胞因子， 抗体和纳米词），甚至病毒和细胞。这是HPF-X将允许冷冻维特里化细胞，组织 样品或整个小生物在与配体进行模拟后的一系列时间点以后 用纳米级成像技术（例如电子显微镜和超分辨率光学）询问 显微镜。这种方法将允许使用纳米级空间捕获配体触发的细胞过程 分辨率和临时分辨率<50 ms。该技术的生物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。虽然 从根本上讲，需要新的HPF仪器设计才能允许缓冲交换，这是我们广泛的初步数据 确认可行性。在AIM 1中，我们将开发一个与缓冲区交换兼容的高压室， 冷冻验证并表征其性能。在AIM 2中，我们将开发一种用于时间分辨的冷冻的方法 使用金标准的生物样品冷却和验证系统。 HPF-X的开发是紧急情况 鉴于纳米级生物成像的冒险，技术机会。重要的是，这项工作超出了当前 由于所有可用的HPF设备都是商业化的，方法是冷冻侵入性领域中的方法开发制度。我们的 定制的HPF-X仪器将允许其他人完全控制，多功能性以及易于采用和修改 研究人员根据他们的项目需求，这是无法通过现成的HPF工具来实现的。