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.

项目概要摘要 配体触发事件对于神经科学、内分泌学、病毒学、免疫学、 然而，刺激后的分子和超微结构变化很难确定。 可视化，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（衍射极限）和标记约束（没有抗体访问或 在活细胞中清洗），而纳米级成像方法要么缺乏时间分辨率来捕获快速动态 （例如，超分辨率光学显微镜）或与活细胞成像完全不兼容（例如，标准或 鉴于这些限制，时间分辨冷冻玻璃化。 该方法非常适合通过冷冻样品在刺激后经过规定的等待时间后捕获细胞过程 成像前处于无定形冰状态的高压冷冻（HPF）通常用于此目的。 因为与大气中的低温骤降相比，其样品厚度限制较宽松（<300 µm） 压力（<10 µm）但是，目前还没有与时间分辨缓冲液交换兼容的 HPF 设备。 为此，我们将开发 HPF-X——一种具有时间分辨缓冲交换能力的 HPF 设备。 冷冻玻璃化之前的缓冲液交换将允许用各种生物和化学物质刺激样品。 药物制剂，包括离子、小分子、肽和蛋白质（例如激素、细胞因子、 因此，HPF-X 将允许冷冻玻璃化细胞、组织。 样品或整个小生物体在用配体刺激后的一系列时间点进行后续分析 使用电子显微镜和超分辨率光学等纳米级成像技术进行询问 这种方法将允许以纳米级空间捕获配体触发的细胞过程。 该技术的分辨率和时间分辨率<50 ms。 蛋白质-蛋白质相互作用、翻译后修饰和蛋白质-膜动力学。 需要全新的 HPF 仪器设计来允许缓冲液交换，我们广泛的初步数据 在目标 1 中，我们将开发一个与缓冲液交换兼容的高压室。 在目标 2 中，我们将开发一种时间分辨冷冻玻璃化方法。 使用金标准生物样本冷却和验证系统是一项新兴的工作。 重要的是，这项工作超越了当前的水平。 冷冻玻璃化领域的方法开发体系，因为我们所有可用的 HPF 设备都是商业化的。 定制的 HPF-X 仪器将允许完全控制、多功能性以及易于其他人采用和修改 研究人员可以根据自己的项目需求进行定制，这是现成的 HPF 仪器无法实现的。