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.

项目摘要 已经证明三维（3D）细胞培养模型的行为与动物模型更相似 比平池单层。这些人类芯片类型测定的高度相关性是至关重要的 加速药物开发。为了有效图像3D样品，慢单点激光扫描显微镜 正在用基于摄像机的光片显微镜取代其出色的成像速度和降低的光 接触。虽然光片显微镜已成功应用于较大的图像动态过程 诸如果蝇，斑马鱼和秀丽隐杆线虫胚胎中的细胞迁移之类的鳞片，解决动力学 分子水平大多被忽略了。但是，测量生物分子动力学对于 了解细胞信号，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的相互作用 - 可以成为新药靶标的机制 包括免疫治疗药。因此，我们建议开发新的方法来量化分子/粒子 带有光片成像的三维细胞培养模型和组织运动。这个高风险/高 奖励提案将量身定制光片成像以研究具有高空间时间分辨率的蜂窝界面 并利用二维对相关分析来绘制在那些生物分子的路径 接口。 在AIM 1中，我们将使用快速光束扫描，转向和重新聚焦以产生倾斜/倾斜的光线 量身定制的床单。使用基于微龙的自适应的一个或几个复杂平面的成像 检测路径中的光学功能将使我们能够以频率z的速度录制数据速度要高得多 堆栈完成了许多飞机。总体可行性是通过先前使用光束扫描，转向的指示 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AIM 2中，我们将在存在障碍物或障碍的情况下研究分子动力学的空间组织 细胞界面。为了揭示单个像素分辨率的障碍，我们最近建议 维二对相关函数（2D-PCF）方法，但到目前为止，该方法成功地应用了 缺乏3D细胞培养模型。因此，我们打算用光证明这种新策略的有效性 在细胞细胞接触/相互作用的挑战案例中，薄板显微镜。在许多生物医学研究中 涉及细胞 - 细胞接触，膜受体是感兴趣的重点。因此，我们将利用一个模型 天然杀伤细胞与目标癌细胞相互作用以发展我们的方法。 我们的目标是使研究人员能够有效研究分子上3D标本中的细胞相互作用 水平，这对于开发创新免疫疗法方法尤其重要，因为 例如，治疗癌症。