Fluorescence Fluctuation Spectroscopy with Light Sheet Microscopy

项目总结 三维(3D)细胞培养模型已被证明其行为更类似于动物模型 而不是平坦的细胞单层。这些芯片上人类类型的高度生理相关性是关键 加快药品研发。为了高效地对3D样品成像，慢速单点激光扫描显微镜 正在被基于相机的光片显微镜所取代，因为它具有更高的成像速度和更少的光线 曝光。虽然光学薄片显微镜已经成功地应用于对更大尺寸的 在果蝇、斑马鱼和线虫胚胎中的细胞迁移等尺度，解析动态 分子水平的研究大多被忽视。然而，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， 细胞如何参与与周围细胞的相互作用--可能成为新药靶点的机制 包括免疫疗法。因此，我们建议开发新的方法来量化分子/粒子。 光片成像三维细胞培养模型和组织中的运动。这种高风险/高风险 奖励计划将定制光片成像，以研究具有高时空分辨率的细胞界面 并利用二维对相关分析来绘制生物分子在 接口。 在目标1中，我们将使用快速光束扫描、转向和重新聚焦来产生倾斜/倾斜和弯曲的光 为蜂窝接口量身定做的床单。基于微镜的自适应算法对一个或几个复杂平面的成像 检测路径中的光学元件将允许我们以比传统z更高的速率记录数据 由许多飞机组成的堆栈。总体可行性由以前使用的波束扫描、转向 以及使用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，我们将研究在存在障碍或障碍物的情况下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建议： 维对相关函数(2D-PCF)方法，但到目前为止，该方法成功地应用于 目前尚缺乏3D细胞培养模型。因此，我们打算用LIGH证明这一新战略的有效性 在更具挑战性的细胞-细胞接触/相互作用的情况下的薄片显微镜。在许多生物医学研究中 涉及细胞与细胞的接触，膜受体是人们关注的焦点。因此，我们将利用一个模型 自然杀伤细胞与靶癌细胞相互作用，以开发我们的方法。 我们的目标是使研究人员能够有效地研究分子上3D样本中的细胞相互作用 水平，这对开发创新的免疫治疗方法特别重要，对于 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.