Zhang Yongqing, Deng Xinyu, Wang Siming, Zhou Wenyu, Wu Zhengyan, Tang Xiaobin, Lee Hyeon Jeong, Zhang Delong
Zhejiang Key Laboratory of Micro-nano Quantum Chips and Quantum Control, School of Physics, Zhejiang University, Hangzhou, 310027, China.
College of Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou, 310058, China.
Angew Chem Int Ed Engl. 2025 Aug 4;64(32):e202505038. doi: 10.1002/anie.202505038. Epub 2025 Jun 8.
Visualizing cholesterol dynamics in living systems in situ remains a fundamental challenge in biomedical imaging. Although fluorescence microscopy requires bulky tags that perturb small molecule behavior, stimulated Raman scattering (SRS) microscopy enables label-free detection of CH-rich molecules. However, conventional SRS probes only polarized Raman components, limiting molecular specificity by seemingly overlapped peaks. Here, we extend SRS microscopy to achieve rapid, comprehensive detection of Raman tensor through quadrature phase-shifted polarization SRS (QP-SRS) microscopy. This technique exploits the underlying molecular signatures by detecting both polarized and depolarized components of third-order nonlinear susceptibility χ that originates from molecular structural features. We adopt a specialized optical delay line that rapidly alternates between parallel- and perpendicular-polarization states. QP-SRS enables unprecedented distinction of similar molecular species in complex mixtures, demonstrating approximately 10× enhancement in chemical specificity and 5× improvement in analytical accuracy. This enhanced sensitivity enables real-time monitoring of lipid dynamics in living C. elegans and reveals component heterogeneity and morphological changes of LD in NAFLD livers. QP-SRS creates new opportunities for investigating cholesterol-dependent biological processes in their native environment, with broad potential for chemical imaging with enhanced molecular specificity.
在生物医学成像中，原位可视化活体细胞系统中的胆固醇动态变化仍然是一项根本性挑战。尽管荧光显微镜需要使用会干扰小分子行为的大型标记物，但受激拉曼散射（SRS）显微镜能够对富含CH的分子进行无标记检测。然而，传统的SRS探针仅能检测偏振拉曼分量，由于峰形看似重叠，限制了分子特异性。在此，我们通过正交相移偏振SRS（QP-SRS）显微镜扩展了SRS显微镜技术，以实现对拉曼张量的快速、全面检测。该技术通过检测源自分子结构特征的三阶非线性极化率χ的偏振和非偏振分量，利用潜在的分子特征。我们采用了一种专门的光学延迟线，可在平行偏振态和垂直偏振态之间快速交替。QP-SRS能够以前所未有的方式区分复杂混合物中相似的分子种类，化学特异性提高了约10倍，分析精度提高了5倍。这种增强的灵敏度能够实时监测活秀丽隐杆线虫中的脂质动态变化，并揭示非酒精性脂肪性肝病（NAFLD）肝脏中脂滴（LD）的成分异质性和形态变化。QP-SRS为在自然环境中研究胆固醇依赖性生物过程创造了新机会，在增强分子特异性的化学成像方面具有广阔潜力。
