基于复杂介质波前校正的活体动物内细胞的实时捕获与成像

Direct imaging and manipulation of cells in living animals as well as measurement in situ are highly desirable but challenging for biomedical research and clinical diagnosis. Although microscopic objects can be readily manipulated at the cellular level, additional physiological insight is likely to be gained by manipulation of cells in vivo. In-vivo manipulation techniques can enable to directly study the structures and functions of the cells in the natural environment and analyze the interaction between cells and tissues, which will further provide insights into thrombogenesis, metastases of tumor, etc. Recently, the optical tweezers technique has enabled the trapping and manipulation of red blood cells within capillaries in living mice, which expands the new area of optical tweezers for in-vivo research. And a non-contact micro-operation has been managed to clear a blocked microvessel. However, all these studies have so far been limited to the superficial layer of tissue because most biological tissues are highly scattering and the light cannot go deeper into the tissues. So far, the depth of cell trapping in vivo is limited to approximately 50μm with focused Gaussian beams and 100μm with Bessel beams, respectively. However, optical manipulation within deeper tissues has not been realized now but is highly desireable..In this project, we aims to realize optical trapping and measurement of the blood cells in capillaries of millimetric depth within the tissues. To gain this ends, we will focus our research on the dynamic wavefront shaping within complex media, performing in situ detection, investigating how to modulate the light propagation in biological tissues, constructing a high-speed wavefront shaping system with real-time feedback, realizing laser focusing into deep tissues, etc. Furthermore, the quantitative methods for characterization of the trapped cells in living animals will be investigated too. The developed techniques can also be exploited for imaging of single label-free cells in vivo, and the actualization of this research will have great potential to facilitate translation of basic research to the clinic.

活体动物内成像和捕获对研究体内细胞之间以及细胞与组织之间的相互作用、揭示血栓形成机制、肿瘤细胞转移等临床医学研究具有重要意义。申请人于2013年首次利用光镊成功操控活体动物内细胞，开辟了活体动物内光学操控新领域。然而由于激光在生物组织中传播时组织对激光强散射而消弱光的穿透深度，影响体内更深位置光阱的形成。目前贝塞尔光镊可达百微米深度，体内更深位置的光捕获成为活体研究急需发展的技术领域。本项目以实现活体内毫米深度的捕获和测量为目标。基于动态散射介质的波前校正技术，研究以活体组织内光传输特性，通过散射介质内部光信号的原位探测，结合实时反馈的高速空间光调制技术，实现激光在深度组织内部的会聚，应用于血管内捕获细胞及其位移和力等参数测量。其中实时动态波前校正理论和实验成果直接推广应用于活体内单个非标记细胞的成像。本项目将实现活体内毫米深度细胞操控和成像，为活体深度组织内细胞的定量研究提供得力的工具。

动物活体内的细胞捕获和成像具有重要的生物医学基础研究和临床应用前景，然而对深度组织内细胞进行光捕获和成像受限于组织的光散射，成为该领域发展的瓶颈。我们提出了利用散射介质波前调制技术解决生物组织的光散射问题。. 项目执行期间，我们系统地开展了光通过不同厚度散射介质光场性质，不同光束经过散射介质传输特性和动态散射波前调制的理论和实验研究，提出了抗散射光学聚焦和成像的系列方法和实验技术。如测量散射介质时间变化相关系数分析，表征散射样品的动态变化特征时间，建立了多层随机相位叠加稳态化的散射介质物理模型。研究透过散射介质的光场强度、相位和偏振同时调控原理，提出了基于矢量传输矩阵模型的光场多维度调制方案。建立了散射介质光场调制物理模型，开发了散射相关矩阵的信息恢复技术，在强散射条件下实现了光场信息的精确传输，实现了光束经过高散射介质氧化锌沉积物（厚度约80μm）后，在百微米空间的三维光学聚焦和成像，以及同时生成多点聚焦和成像。. 建立了“斑马鱼微注射操控系统”。利用磁性微粒及其热效应，将其注射到活体内作为光束聚焦的引导靶标，有效解决了散射介质内光信号的原位探测。研究锁相原理同时进行标定和测量的方法，解决活体内实时动态环境下光镊力的标定和测量的难题。提出‘光学围栏’法解决了动物活体内细胞密集环境下对单个细胞操控技术。引入抗散射的单像显微成像技术，研究单像素相位成像和相衬成像方法，应用于流动细胞成像。. 基于复杂介质波前调制的理论和实验研究，以及若干个关键技术的突破，我们逐步构建了基于复杂介质波前校正的光学捕获实验平台。研究成果有力地推进了活体光镊技术领域的发展，也收获了交叉学科的人才成长。散射介质光场调控的方法和技术成果有望在生物医学成像、光通信等领域产生重要的应用价值。