Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy
Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy
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DOI：
10.1073/pnas.1813044116
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发表时间：
2019-04-02
影响因子：
11.1
通讯作者：
Min, Wei
中科院分区：
文献类型：
--
作者：
Wei, Mian;Shi, Lingyan;Min, Wei
利用光学显微镜对组织结构进行三维可视化有助于理解生物功能。然而，由于严重的光散射，光学显微镜在组织穿透方面受到限制。最近，出现了一系列组织清除技术，以显著扩展荧光成像的深度。受这些进展的启发，我们开发了一种体积化学成像技术，将拉曼定制的组织清除与受激拉曼散射(SRS)显微镜相结合。与标准SRS相比，清除增强SRS的深度增加了10倍以上。基于提取的蛋白质和脂肪的空间分布，我们的方法揭示了肿瘤球体、小鼠脑组织和肿瘤异种移植的复杂的3D组织。我们进一步发展了多光谱SRS图像的体积相量分析，用于3D中特定化学成分的聚类和分割。此外，超越了传统的无标记范例，我们展示了代谢体积化学成像，这使我们能够同时绘制出胶质母细胞瘤中蛋白质和脂肪合成的代谢活动。总之，这些结果支持体积化学成像作为一种有价值的工具，在不同的生物环境中阐明全面的3D结构、组成和功能，补充流行的体积荧光显微镜。
Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.