Fluorescence Enhanced Photothermal Infrared Spectroscopy (FE-PTIR)-breakthrough for simultaneous fluorescence microscopy and sub-micron IR spectroscopy

本第一阶段提案旨在开发和证明荧光增强光热红外的可行性 （FE-PTIR）成像和光谱学。拟议的FE-PTIR将使用荧光显微镜来绘制 荧光标记的细胞和组织区域，然后使用 光热红外光谱荧光显微镜是生物研究中的基石技术， 敏感和高度特异性的细胞和组织内的目标生物分子的映射，但它不提供信息 这些分子的化学结构。红外光谱可以提供丰富的化学结构分析 并已用于生命科学研究，以研究组织分类、药物/组织相互作用、神经退行性疾病 癌症研究和其他领域。然而，传统的红外光谱法在其应用上具有根本的限制。 空间分辨率（即它可以分析的物体的大小）约为10微米，类似于一个物体的大小。 平均生物细胞。因此，传统的红外光谱法对于许多生物医学领域来说是非常有限的。 其中感兴趣的结构小于细胞的尺寸的应用。 FE-PTIR技术将克服荧光显微镜和红外光谱的局限性 为了提供目标生物分子的高度特异性映射沿着这些分子的化学结构分析， 具有与荧光显微镜相同的空间分辨率。这个项目将通过使用一本小说来实现这一突破 光学光热红外光谱的形式，以测量荧光标记的区域的红外光谱， sample.具体地，FE-PTIR技术将用红外激光源照射样品，所述红外激光源可以被调谐以 激发感兴趣的样品的分子振动。一个独立的紫外/可见光光源将用于两项工作：（1） 在样品的荧光标记区域中激发荧光发射;以及（2）测量局部加热， 吸收红外线辐射。通过测量从不同区域发射的荧光强度， 样品，可以绘制荧光标记的生物分子的分布。然后通过测量 由于局部IR诱导加热而从样品收集的UV/可见光的量， 产生相同位置和相同空间分辨率的红外吸收光谱。红外吸收 然后，光谱可用于分析样品的荧光标记区域的化学结构。这 该项目与NIH的目标非常一致，因为它包含了国家生物医学成像研究所的几个关键目标 和生物工程，包括光学成像和光谱学，红外成像，共聚焦显微镜，和多模态 显像FE-PTIR将是非常有用的，例如，在定位特定的蛋白质与荧光显微镜，然后 使用光热红外光谱分析它们的结构，例如蛋白质是如何折叠的。蛋白 错误折叠是许多神经退行性疾病（例如阿尔茨海默病）的根本原因，FE-PTIR将提供新的见解。 展示FE-PTIR技术将使一种新的多模态显微镜具有亚细胞分辨率， 对生物医学研究，包括神经退行性疾病和抗菌素耐药性研究具有深远的益处。

This Phase I proposal aims to develop and demonstrate the feasibility of Fluorescence Enhanced Photothermal Infrared (FE-PTIR) imaging and spectroscopy. The proposed FE-PTIR will use fluorescence microscopy to map the distribution of fluorescently labeled regions of cells and tissue and then provide chemical structural analysis of the labeled regions using photothermal infrared spectroscopy. Fluorescence microscopy is a cornerstone technique in biological research, allowing sensitive and highly specific mapping of target biomolecules within cells and tissue, but it does not provide information about the chemical structure of those molecules. Infrared spectroscopy can provide rich analysis of chemical structure and has been used in life sciences research to study tissue classification, drug/tissue interaction, neurodegenerative diseases, cancer research and other areas. Conventional infrared spectroscopy, however, has a fundamental limit on its spatial resolution (i.e. roughly how small an object it can analyze) of around 10 micrometers, similar to the size of an average biological cell. Thus conventional infrared spectroscopy has been extremely limited for many biomedical applications where the structures of interest are smaller than the size of a cell. The proposed FE-PTIR technique will overcome the limitation of both fluorescence microscopy and infrared spectroscopy to provide highly specific mapping of target biomolecules along with chemical structural analysis of those molecules, both with the same spatial resolution as fluorescence microscopy. This project will achieve this breakthrough by using a novel form of optical photothermal infrared spectroscopy to measure infrared spectra of fluorescently labeled regions of a sample. Specifically, the FE-PTIR technique will illuminate a sample with an infrared laser source that can be tuned to excite molecular vibrations a sample of interest. A separate ultraviolet/visible light source will be used for two jobs: (1) to excite fluorescent emission in fluorescently labeled regions of the sample; and (2) measure a localized heating resulting from absorption of infrared radiation. By measuring the intensity of fluorescent light emitted from different regions of the sample, it is possible to map the distribution of fluorescently labeled biomolecules. Then by measuring subtle changes in the amount of UV/visible light collected from the sample resulting from the local IR-induced heating, it is possible to generate infrared absorption spectra of the same locations and with the same spatial resolution. The infrared absorption spectrum can then be used to analyze the chemical structure of the fluorescently labeled regions of the sample. This project is well aligned with NIH goals as it incorporates several key thrusts of the National Institute of Biomedical Imaging and Bioengineering, including optical imaging and spectroscopy, IR imaging, confocal microscopy, and multimodal imaging. FE-PTIR will be extremely useful for example in localizing specific proteins with fluorescence microscopy and then analyzing using photothermal IR spectroscopy to analyze their structure, for example how the protein is folded. Protein misfolding is a root cause of many neurodegenerative diseases (e.g. Alzheimer’s) and FE-PTIR will offer new insights. Demonstrating the FE-PTIR technology will enable a new multimodal microscope with sub-cellular resolution that will offer profound benefits for biomedical research including neurodegenerative diseases and antimicrobial resistance research.