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 配体触发的事件是神经科学、内分泌学、病毒学、免疫学 和药理学。然而，分子和超微结构的变化，以下的刺激是困难的， 因为它们涉及蛋白质和膜的快速纳米级运动和修饰。国家 现有技术不足以捕获这些时空变化。例如，活体荧光 成像受到空间分辨率（衍射极限）和标记约束（没有抗体进入或 在活细胞中洗涤），而纳米级成像方法要么缺乏时间分辨率以捕获快速动态 (e.g.,超分辨率光学显微术）或与活细胞成像完全不兼容（例如，标准或 冷冻电子显微术;膨胀显微术）。鉴于这些限制，时间分辨冷冻玻璃化 方法是通过冷冻样本在刺激后经过规定的等待时间后捕获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）常用于此目的 由于与大气压下的低温插入相比，其放宽了样品厚度限制（<300 µm） 压力（<10 µm）。然而，与时间分辨缓冲区交换兼容的HPF设备目前还不 存在.为此，我们将开发HPF-X -一种具有时间分辨缓冲液交换能力的HPF设备 在低温玻璃化之前。缓冲液交换将允许用各种生物和化学物质刺激样品。 药理学试剂包括离子、小分子、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 在用配体刺激后的一系列时间点，将样品或整个小生物体用于随后的 利用纳米级成像技术如电子显微镜和超分辨率光学 显微镜这种方法将允许捕获配体触发的细胞过程， 分辨率和时间分辨率<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 需要一种全新的HPF仪器设计，以允许缓冲液交换，我们的大量初步数据表明， 证实了可行性。在目标1中，我们将开发与缓冲液交换兼容的高压室， 低温玻璃化并表征其性能。在目标2中，我们将开发一种时间分辨冷冻的方法， 冷却并使用金标准生物样品验证系统。HPF-X的发展是一个紧急的 纳米生物成像技术的出现带来了巨大的技术机遇。重要的是，这项工作超越了目前的 这是因为所有可用的HPF设备都是商业化的。我们 定制的HPF-X仪器将允许完全控制，多功能性，易于采用和修改的其他 研究人员根据他们的项目需要，这是无法实现与现成的HPF仪器。