Photothermal Spectroscopy Corporation, Santa Barbara, California93101, United States.
Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, California93106, United States.
J Med Chem. 2023 Feb 23;66(4):2542-2549. doi: 10.1021/acs.jmedchem.2c01359. Epub 2023 Jan 4.
Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates.
红外光谱成像是广泛用于生物分子结构的可视化，和技术，如光光热红外（OPTIR）微光谱可以实现<500nm 的空间分辨率。然而，这些方法缺乏对特定细胞类型和细胞成分的特异性，因此不能作为一种独立的技术来评估它们的特性。在这里，我们开发了一种新的工具，荧光引导光热红外显微镜，同时利用荧光成像和 OPTIR 进行荧光引导的红外光谱分析。这种新方法超过了红外显微镜的衍射极限，并允许在组织和单个细胞中直接对特定蛋白质进行结构分析。本文所述的实验使用荧光快速定位组织或神经元培养物中的淀粉样蛋白，从而引导 OPTIR 测量以评估亚细胞水平的淀粉样结构。我们相信，这种新方法将是红外光谱的一个有价值的补充，为复杂系统中结构改变的蛋白质聚集体的研究提供了细胞特异性的测量。
