Development of a fast scanning, extended field-of-view multiphoton microscope for clinical skin imaging

摘要 研究哺乳动物的免疫细胞及其与组织的原位相互作用对于理解 它们如何调控从伤口愈合到自身免疫性疾病的启动到癌症和 设计更好的治疗策略来治疗这些流行的疾病。活体多光子显微镜 (MPM)结合丰富的荧光报告小鼠模型和体内细胞和组织标记 技术已经使在皮肤和组织的亚细胞水平上可视化免疫细胞-组织相互作用成为可能 其他器官。然而，人类皮肤的结构和免疫环境存在显著差异 将这些发现的可译性限制在人类皮肤免疫反应上。我们小组最近 开发了一种快速大面积多光子外窥镜(FLAMP)，这是一种独特的成像平台，优化了 临床皮肤成像可快速生成显微分辨率(0.5-0.5)的宏观图像(毫米级到厘米级) 1微米)，基于无标记分子对比度(荧光强度和寿命)。在此应用程序中，我们利用 我们在MPM技术开发和临床成像方面的丰富经验为400多名患者提供服务 在过去的几年里开发了第一个基于MPM的临床设备(IFLAME)作为研究成像工具 优化并致力于体内免疫细胞群体及其动力学的无标记成像 人的皮肤。在目标1中，我们开发了iFLAME作为一种临床研究工具，用于高效的体内无标记成像 人类皮肤中的真皮细胞种群及其动态。这项工作涉及到检测和开发 能够实现快速荧光寿命检测的分析方法以及光学和计算方法 以及自动测量细胞形态和代谢特征所需的分析。在……里面 目的2，我们通过展示正常人体内免疫细胞的特性来验证iFLAME的性能 和发炎的人类皮肤。在目标3中，我们开发了定量形态和代谢MPM成像 在监测伤口的情况下评估人体皮肤免疫渗透及其动力学的终点 治愈。这项工作代表了第一次尝试使用MPM的内在来源的对比度来成像、识别和 根据人体皮肤的光学特征和迁移行为，对体内的关键免疫细胞进行量化。我们的 长期目标是开发iFLAME作为临床研究工具，用于快速、无标记地对免疫细胞进行成像 基于细胞形态和代谢成像终点的皮肤。这些可以用来更好地理解， 评估和优化伤口愈合、自身免疫性皮肤病和治疗反应。

Summary The study of mammalian immune cells and their interactions with tissue in situ is critical for understanding how they regulate processes ranging from wound healing to autoimmune disease initiation to cancer and for designing better therapeutic strategies to treat these prevalent conditions. Intravital multiphoton microscopy (MPM) combined with a rich repertoire of fluorescent reporter mouse models and in vivo cell and tissue labeling techniques have made it possible to visualize immune cell-tissue interactions at a subcellular level in skin and other organs. However, there are significant differences in the structure and immune milieu of human skin that limits the translatability of these findings to the human cutaneous immune response. Our group has recently developed a fast large area multiphoton exoscope (FLAME), a unique imaging platform optimized for efficient clinical skin imaging to rapidly generate macroscopic images (mm to cm-scale) with microscopic resolution (0.5- 1µm) based on label-free molecular contrast (fluorescence intensity and lifetime). In this application, we leverage our extensive experience in MPM technology development and clinical imaging of more than 400 patients over the past several years to develop the first MPM-based clinical device (iFLAME) as a research imaging tool optimized for, and dedicated to, in vivo label-free imaging of immune cell populations and their dynamics in human skin. In Aim 1, we develop iFLAME as a clinical research tool for efficient in vivo label-free imaging of dermal cell populations and their dynamics in human skin. This work involves development of detection and analytic approaches as well as optical and computational methods to enable rapid fluorescence lifetime detection and analysis necessary to automate measurements of the cellular morphological and metabolic signatures. In Aim 2, we validate iFLAME performance by demonstrating in vivo characterization of immune cells in normal and inflamed human skin. In Aim 3, we develop quantitative morphological and metabolic MPM imaging endpoints to assess immune infiltrates and their dynamics in human skin in the context of monitoring wound healing. This work represents the first attempt to use intrinsic sources of MPM contrast to image, identify, and quantify key immune cells in human skin in vivo based on their optical signatures and migratory behavior. Our long-term goal is to develop iFLAME as a clinical research tool for rapid, label-free imaging of immune cells in skin based on cellular morphologic and metabolic imaging endpoints. These can be used to better understand, evaluate and optimize wound healing, autoimmune skin diseases and therapeutic responses.