Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples
Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples
复制标题
DOI：
10.3390/mps2020051
复制
发表时间：
2019-06-01
影响因子：
2.4
通讯作者：
Pavone, Francesco Saverio
中科院分区：
文献类型：
--
作者：
Marchetti, Marco;Baria, Enrico;Pavone, Francesco Saverio
现代光学提供了多种无标记显微和光谱解决方案，可用于生物组织的成像和病理评估。通过快速且无标记的技术获得相似的形态学和生化信息的可能性是非常可取的，但是没有单一的光学模式能够获得组织学和免疫组织化学分析提供的所有信息。集成多模态成像提供了以无标记方式整合形态学与功能化学信息的可能性，用多种特定对比机制补充了简单观察。在这里，我们开发了一个定制的激光扫描显微平台，将共焦拉曼光谱与多模态非线性成像相结合，包括相干反斯托克斯拉曼散射、二次谐波产生、双光子激发荧光和荧光寿命成像显微镜。该实验装置能够对标本进行高分辨率形态成像，同时还提供有关分子组织、功能行为和分子指纹的具体信息。该系统在受尿路上皮癌和动脉粥样硬化影响的离体组织分析中成功进行了测试，使我们能够对所研究的样本进行多模式表征。我们的结果显示了原理验证，证明了所提出的多模式方法的潜力，该方法可用于广泛的生物和生物医学应用。
Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging offers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues affected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications.