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

第二阶段项目旨在开发和商业化荧光增强光热红外（FE-PTIR）成像 2、光谱学拟议的FE-PTIR将使用荧光显微镜来绘制荧光标记的 3个区域的细胞和组织，然后使用光热红外提供标记区域的化学结构分析 4光谱学。荧光显微镜是生物学研究中的基石技术，可以灵敏地绘制 5特异性靶向细胞和组织内的生物分子，但它不提供有关其分子的信息。 6结构。红外光谱技术可以提供丰富的分子结构分析，已广泛应用于生命科学领域 7.研究组织分类、药物/组织相互作用、神经退行性疾病、癌症等领域。 8然而，传统的红外光谱学具有基本的空间分辨率限制（即，它可以测量物体的大小）。 9分析）约10微米，类似于平均生物细胞的大小。因此，常规的IR光谱具有 对于许多生物医学应用，其中感兴趣的结构小于细胞，这种方法受到极大的限制。 11 FE-PTIR技术用UV/可见光照射荧光标记的样品，其产生荧光标记。 图12示出了来自样品中的荧光标记分子的发射。可调谐红外激光源也照射样品， 如果IR激光器被调谐到激发样品中的分子振动的波长，则在样品中引起局部加热。 14样品。使用照相机或其他敏感的光电探测器用于记录来自不同区域的荧光发射 15的样品生成荧光标记的生物分子的分布图。该提案的一个关键创新之处在于 16是认识到普通荧光团具有高度依赖于温度的发射效率。因此 当样品也用对应于分子振动的波长的红外光照射时， 来自样品的IR吸收的加热导致荧光发射的显著变化。记录发射 作为样品位置或IR波长的函数的变化产生IR吸收图像和IR吸收光谱， 20分别。第一阶段的研究显示了以下关键进展：（1）FE-PTIR的目标定位能力 21个具有荧光的生物分子，并分析目标分子的分子结构;（2）实现亚微米 22空间分辨率的荧光成像和红外光谱;（3）证明了100倍的改善， （4）FE-PTIR技术在神经退行性疾病相关蛋白质错误折叠研究中的应用 （5）在单个细菌和活癌细胞上以亚细胞分辨率证明了FE-PTIR。这个项目是 25与NIH的目标保持一致，因为它结合了国家生物医学成像研究所的几个关键目标， 26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成像。FE- 27 PTIR将非常有用，例如在分析荧光的分子结构/折叠/聚集方面。 28种蛋白质蛋白质错误折叠/聚集是许多神经退行性疾病（例如阿尔茨海默病）的根本原因。 第二阶段项目的完成将导致一种新的多模态显微镜的商业化， 生物医学研究的30个深远的好处，包括神经退行性疾病和抗菌素耐药性研究。

1 This Phase II project aims to develop and commercialize Fluorescence Enhanced Photothermal Infrared (FE-PTIR) imaging 2 and spectroscopy. The proposed FE-PTIR will use fluorescence microscopy to map the distribution of fluorescently labeled 3 regions of cells and tissue and then provide chemical structural analysis of the labeled regions using photothermal infrared 4 spectroscopy. Fluorescence microscopy is a cornerstone technique in biological research, allowing sensitive mapping of 5 specifically targeted biomolecules within cells and tissue, but it does not provide information about their molecular 6 structure. Infrared (IR) spectroscopy can provide rich analysis of molecular structure and has been used in life sciences 7 research to study tissue classification, drug/tissue interaction, neurodegenerative diseases, cancer and other areas. 8 Conventional IR spectroscopy, however, has a fundamental spatial resolution limit (i.e. roughly how small an object it can 9 analyze) of around 10 micrometers, similar to the size of an average biological cell. Thus conventional IR spectroscopy has 10 been extremely limited for many biomedical applications where the structures of interest are smaller than a cell. 11 The FE-PTIR technique illuminates a fluorescently labeled sample with UV/visible light which results in fluorescent 12 emission from fluorescently tagged molecules in the sample. A tunable infrared laser source also illuminates the sample, 13 causing localized heating in the sample if the IR laser is tuned to a wavelength that excites molecular vibrations in the 14 sample. Using a camera or other sensitive photodetector is used to record the fluorescent emission from different regions 15 of the sample generates a map of the distribution of fluorescently labeled biomolecules. A key innovation of this proposal 16 was the recognition that common fluorophores have an emission efficiency that is highly temperature dependent. Thus 17 when the sample is also irradiated with infrared light at wavelengths corresponding to molecular vibrations, localized 18 heating from IR absorption by the sample causes a significant change in the fluorescent emission. Recording the emission 19 change as a function of sample position or IR wavelength produces IR absorption images and IR absorption spectra, 20 respectively. Phase I research demonstrated the following key advances: (1) ability of FE-PTIR to map of target 21 biomolecules with fluorescence and analyze the molecular structural of the target molecules; (2) achieve submicron 22 spatial resolution for both fluorescence imaging and infrared spectroscopy; (3) demonstrated a 100X improvement in 23 measurement sensitivity; (4) application of FE-PTIR to study of protein misfolding relevant to neurodegenerative disease 24 research; (5) demonstrated FE-PTIR on individual bacterial and live cancer cells with subcellular resolution. This project is 25 well aligned with NIH goals as it incorporates several key thrusts of the National Institute of Biomedical Imaging and 26 Bioengineering, including optical imaging and spectroscopy, IR imaging, confocal microscopy, and multimodal imaging. FE- 27 PTIR will be extremely useful for example in analyzing the molecular structure/folding/aggregation of fluorescence- 28 localized proteins. Protein misfolding/aggregation is a root cause of many neurodegenerative diseases (e.g. Alzheimer’s). 29 Completion of this Phase II project will lead to the commercialization of a new multimodal microscope that will offer 30 profound benefits for biomedical research including neurodegenerative diseases and antimicrobial resistance research.