Fluorescence Fluctuation Spectroscopy with Light Sheet Microscopy

荧光涨落光谱与光片显微镜

• 批准号: 10242938
• 负责人: ENRICO GRATTON
• 金额: $ 19.63万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242938
• 关键词: 3-Dimensional; Address; Affect; Animal Model; Anisotropy; Behavior; Biological Assay; Caenorhabditis elegans; Cell Culture Techniques; Cell Surface Receptors; Cells; Characteristics; Clinical Trials; Complex; Consumption; Data; Detection; Development; Dose; Drosophila genus; Effectiveness; Embryo; Environment; Fluorescence; Gene Expression; Glass; Goals; Hour; Human; Image; Immunotherapeutic agent; Immunotherapy; Laser Scanning Microscopy; Light; Lighting; Malignant Neoplasms; Maps; Measures; Membrane; Methods; Microscopy; Modeling; Molecular; Motion; Movement; Natural Killer Cells; Optics; Pharmaceutical Preparations; Photobleaching; Physiological; Positioning Attribute; Process; Proteins; Receptor Cell; Research Personnel; Resolution; Sampling; Scanning; Signal Transduction; Specimen; Spectrum Analysis; Speed; Stimulus; Structure; Surface; Technology; Testing; Time; Tissues; Visualization; Work; Zebrafish; adaptive optics; base; cancer cell; cell motility; cellular imaging; drug candidate; drug development; drug discovery; fluorescence imaging; fluorophore; grasp; high reward; high risk; innovation; intercellular communication; interest; light curve; light scattering; mechanotransduction; millisecond; monolayer; neglect; new therapeutic target; novel; novel strategies; particle; premature; receptor; response; spatiotemporal; three dimensional cell culture; tissue/cell culture; two-dimensional

PROJECT SUMMARY Three-dimensional (3D) cell culture models have been demonstrated to behave more similar to animal models than flat cell monolayers. The high physiological relevance of those human-on-a-chip type assays is key to accelerate drug development. To efficiently image 3D specimen, slow single point laser scanning microscopy is being replaced by camera-based light sheet microscopy for its superior imaging speed and reduced light exposure. While light sheet microscopy has been successfully applied to image dynamic processes on larger scales such as cell migration in Drosophila, zebrafish, and C. elegans embryos, resolving dynamics on the molecular level has been mostly neglected. However, measuring biomolecular dynamics is very important to understand cell signaling, cellular responses, spatial organization of cell surface receptors, and, especially, how cells engage in interactions with surrounding cells – mechanisms that can be targets of new drugs including immunotherapeutics. Hence, we propose to develop novel approaches to quantify molecule/particle movement in three-dimensional cell culture models and tissues with light sheet imaging. This high risk/high reward proposal will tailor light sheet imaging to study cellular interfaces with high spatiotemporal resolution and leverage two-dimensional pair correlation analysis to map the paths of biomolecules taken at those interfaces. In aim 1, we will use fast beam scanning, steering, and refocusing to generate tipped/tilted and curved light sheets tailored to cellular interfaces. Imaging of one or a few complex planes using micromirror-based adaptive optics in the detection path will allow us to record data at a much higher rate than possible with conventional z stacks comprised of many planes. Overall feasibility is indicated by previous use of beam scanning, steering and refocusing to track single particles on the millisecond timescale with the orbital tracking approach. In aim 2, we will study the spatial organization of molecule dynamics in the presence of barriers or obstacles at cellular interfaces. To reveal those barriers with single pixel resolution, we recently suggested the two- dimensional pair correlation function (2D-pCF) approach but, so far, a successful application of this method to 3D cell culture models is lacking. Hence, we intend to prove the effectiveness of this new strategy with light sheet microscopy in the more challenging case of cell-cell contacts/interactions. In many biomedical studies involving cell-cell contacts, membrane receptors are the focus of interest. Therefore, we will utilize a model of natural killer cells interacting with target cancer cells to develop our approach. Our goal is to enable researchers to efficiently study cellular interactions in 3D specimen on the molecular level, which are especially important for the development of innovative immunotherapy approaches, for example, to treat cancers.

项目概要 三维 (3D) 细胞培养模型已被证明与动物模型更加相似 与扁平细胞单层相比，这些人类芯片类型测定的高生理相关性是关键。 为了有效地对 3D 样本进行成像，请使用慢速单点激光扫描显微镜来加速药物开发。 因其卓越的成像速度和减少的光线而被基于相机的光片显微镜所取代 而光片显微镜已成功应用于较大图像的动态处理。 果蝇、斑马鱼和线虫胚胎中的细胞迁移等尺度，解决了 分子水平大多被忽视，然而，测量生物分子动力学非常重要。 了解细胞信号传导、细胞反应、细胞表面受体的空间组织，尤其是 细胞如何与周围细胞相互作用——可以成为新药靶点的机制 因此，我们建议开发新的方法来量化分子/颗粒。 三维细胞培养模型和组织中的光片成像运动。 奖励提案将定制光片成像以研究具有高时空分辨率的细胞界面 并利用二维配对相关分析来绘制生物分子在这些路径上的路径 接口。 在目标 1 中，我们将使用快速光束扫描、转向和重新聚焦来生成倾斜/倾斜和弯曲的光 使用基于微镜的自适应对一个或几个复杂平面进行定制的薄片。 检测路径中的光学器件将使我们能够以比传统 z 更高的速率记录数据 由许多平面组成的堆栈通过先前使用的光束扫描、转向表明了总体可行性。 并通过轨道跟踪方法重新聚焦以在毫秒时间尺度上跟踪单个粒子。 在目标 2 中，我们将研究存在障碍物时分子动力学的空间组织 为了揭示单像素分辨率的这些障碍，我们最近提出了两种方法： 维对相关函数 (2D-pC​​F) 方法，但到目前为止，该方法已成功应用于 由于缺乏 3D 细胞培养模型，我们打算用光来证明这种新策略的有效性。 在许多生物医学研究中，细胞与细胞接触/相互作用更具挑战性的情况中使用片状显微镜。 细胞与细胞的接触、膜受体是关注的焦点，因此，我们将利用涉及的模型。 自然杀伤细胞与靶癌细胞相互作用来开发我们的方法。 我们的目标是使研究人员能够在分子水平上有效地研究 3D 样本中的细胞相互作用。 水平，这对于创新免疫治疗方法的开发尤其重要 例如，治疗癌症。