Fluorescence Fluctuation Spectroscopy with Light Sheet Microscopy

项目摘要 已经证明三维（3D）细胞培养模型表现得更类似于动物模型 而不是扁平的细胞单层。这些人芯片型测定的高度生理相关性是 加快药物开发。为了有效地对3D样品进行成像，慢速单点激光扫描显微镜 由于其上级成像速度和减少的光线， exposure.虽然光片显微镜已成功地应用于图像的动态过程中较大的 如果蝇、斑马鱼和C.线虫胚胎，在 分子水平的研究大多被忽略了。然而，测量生物分子动力学对于 了解细胞信号传导，细胞反应，细胞表面受体的空间组织，特别是， 细胞如何与周围细胞相互作用--可能成为新药靶点的机制 包括免疫治疗。因此，我们建议开发新的方法来量化分子/颗粒 在三维细胞培养模型和组织中的运动与光片成像。高风险/高风险 一项奖励提案将调整光片成像，以研究具有高时空分辨率的细胞界面 并利用二维对相关分析来绘制生物分子在那些 接口。 在aim 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 为细胞界面量身定制的片材。使用基于微镜的自适应成像一个或几个复杂平面 检测路径中的光学器件将使我们能够以比传统z 由许多平面组成的堆栈。总体可行性表明，以前使用的光束扫描，转向 并重新聚焦，以轨道跟踪方法在毫秒时间尺度上跟踪单个粒子。 在aim 2中，我们将研究存在障碍物或障碍物时分子动力学的空间组织， 蜂窝接口。为了用单像素分辨率揭示这些障碍，我们最近提出了两个- 二维对相关函数（2D-pCF）方法，但到目前为止，这种方法的成功应用， 缺乏3D细胞培养模型。因此，我们打算证明这种新策略的有效性与光 在细胞-细胞接触/相互作用的更具挑战性的情况下，在许多生物医学研究中 涉及细胞-细胞接触的膜受体是关注的焦点。因此，我们将使用一个模型， 自然杀伤细胞与靶癌细胞相互作用，以发展我们的方法。 我们的目标是使研究人员能够在分子水平上有效地研究3D标本中的细胞相互作用。 水平，这是特别重要的创新免疫治疗方法的发展， 例如，治疗癌症。

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.