相干拉曼散射显微及其活体成像最新研究进展

doi:10.3964/j.issn.1000-0593(2025)07-1801-08

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摘要：活体成像技术具有革命性意义，能够在生物体内实现实时观测，提供与生命过程密切相关的动态信息，对于揭示疾病发生发展机制和评估治疗效果等具有重要作用。相干拉曼散射（CRS）显微成像技术无需引入可能干扰生物分子正常功能的荧光标记物，可直接对生物样品中特定分子进行特异性成像，在活体成像领域展现出显著优势。然而，在实际应用于活体成像时，CRS显微技术仍面临光损伤、成像深度受限和运动伪影等挑战。近年来，随着相关技术的飞速发展，CRS显微成像在克服光损伤难题、突破成像深度限制、消除或减少运动伪影以及实现多模态融合成像等方面取得了诸多突破，并成功应用于人体皮肤以及实验动物模型的大脑、脊柱等部位和体内肿瘤的实时成像监测，有力推动了该技术在活体成像研究和临床应用中的发展。本文全面综述了CRS显微技术在活体成像中的最新研究进展，深入剖析其面临的挑战和相应解决方案，旨在为该技术的持续发展和广泛应用提供参考。克服光损伤的常用方法是通过限制激光功率和积分时间来减少激光在样品中引起的热效应和化学反应；突破成像深度限制的方案包括对皮肤或皮肤附近部位进行成像、结合光学窗口或直接对微创手术暴露的深层组织或器官进行成像、采用AO技术兼顾深度和无创成像以及采用内窥成像技术等；消除或减少运动伪影首先需要以适当的麻醉和固定方式保持生物体静止或减少运动，其次可应用光学窗口和实时运动矫正算法进一步隔绝麻醉样品因呼吸和心跳等生理活动引起的抖动影响，还可以通过设法提高成像速度来减少运动伪影；此外，结合双光子激发荧光和二次谐波产生等多种非线性光学显微成像实现CRS多模态融合成像，能获取更丰富的信息，增强对活体生物样品的分析能力，助力对生物学信息的深入理解。

关键词：相干拉曼散射显微成像；相干反斯托克斯拉曼散射；受激拉曼散射；活体成像

Abstract：In vivo imaging technology has revolutionary significance, enabling real-time observation of living organisms and providing dynamic information directly related to biological processes. This is crucial for understanding disease mechanisms and evaluating treatment effects. Coherent Raman scattering (CRS) microscopy offers the advantage of specific molecular imaging in biological samples without the need for fluorescent probes that might interfere with biomolecular function, making it a promising tool in the field of in vivo imaging. However, CRS microscopy still faces challenges in practical applications in in vivo imaging, including photodamage, limited imaging depth, and motion artifacts. Recent advancements in related technology have led to significant breakthroughs, addressing these challenges by minimizing photodamage, extending imaging depth, eliminating or reducing motion artifacts, and enabling multimodal imaging. In vivo real-time imaging of human skin, brain, and spine in experimental animal models and tumors has driven substantial progress in CRS microscopy, both in in vivo imaging research and clinical applications. This paper offers a comprehensive review of the latest developments in CRS microscopy for in vivo imaging, providing an in-depth analysis of current challenges and their solutions to contribute to this technology's ongoing development and broader application. Common strategies to overcome photodamage involve reducing the thermal effects and chemical reactions induced by the laser in the sample, typically by limiting laser power and integration time. Several approaches have been explored to address the limitation of imaging depth, including imaging superficial tissues such as the skin or areas near the surface, combining optical windows, or directly imaging deeper tissues or organs exposed through minimally invasive surgery. Adaptive optics technology helps balance depth with non-invasive imaging, while endoscopic imaging provides an additional solution. To minimize or eliminate motion artifacts, it is crucial first to keep the organism stationary or reduce movement through appropriate anesthesia and fixation techniques. In addition, optical windows and real-time motion correction algorithms can be employed to mitigate further jitter caused by physiological activities like breathing and heartbeat in anesthetized samples. Increasing imaging speed is another way to reduce motion artifacts. Finally, combining CRS with other nonlinear optical microscopy techniques, such as two-photon excitation fluorescence and second-harmonic generation, enables multimodal imaging, providing richer information, enhancing the analysis of in vivo biological samples, and offering deeper insights into biological processes.

Key words：Coherent Raman scattering microscopy; Coherent anti-Stokes Raman scattering; Stimulated Raman scattering; In vivo imaging

收稿日期: 2025-01-20 修订日期: 2025-03-04

中图分类号:

O657.37

基金资助: 国家重点研发计划课题(2022YFF0706001)，国家自然科学基金项目(62235007，62275165，62175166，62275166，T2421003)，广东省基础与应用基础研究基金项目(2024A1515012567)资助

通讯作者: 林丹樱 E-mail: dylin@szu.edu.cn

作者简介: 李舒琦，女，1998年生，深圳大学物理与光电工程学院硕士研究生 e-mail: 2200453050@email.szu.edu.cn

引用本文:

李舒琦，骆国权，陈钰，于斌，屈军乐，林丹樱. 相干拉曼散射显微及其活体成像最新研究进展[J]. 光谱学与光谱分析, 2025, 45(07): 1801-1808.
LI Shu-qi, LUO Guo-quan, CHEN Yu, YU Bin, QU Jun-le, LIN Dan-ying. Coherent Raman Scattering Microscopy and Its Recent Research Progress in in Vivo Imaging. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2025, 45(07): 1801-1808.

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