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

荧光增强光热红外光谱 (FE-PTIR)——同步荧光显微镜和亚微米红外光谱的突破

• 批准号: 10253663
• 负责人: Craig Prater
• 金额: $ 25.66万
• 依托单位: PHOTOTHERMAL SPECTROSCOPY CORP.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-02 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10253663
• 关键词: Address; Alzheimer' s Disease; Antimicrobial Resistance; Antimicrobial susceptibility; Area; Biochemical; Biocompatible Materials; Biological; Biological Sciences; Biology; Biomedical Research; Boston; Cells; Chemical Structure; Chemicals; Classification; Computer software; Confocal Microscopy; Dependence; Detection; Development; Disease Resistance; Fluorescence; Fluorescence Microscopy; Goals; Heating; Image; In Situ; Individual; Infrared Rays; Label; Lasers; Light; Location; Maps; Measurement; Measures; Microscope; Microscopy; Molecular; Multimodal Imaging; National Institute of Biomedical Imaging and Bioengineering; Neurodegenerative Disorders; Occupations; Optical Instrument; Optics; Performance; Pharmaceutical Preparations; Phase; Physiologic pulse; Plant Roots; Preparation; Proteins; Protocols documentation; Research; Resolution; Sampling; Science; Secondary Protein Structure; Small Business Innovation Research Grant; Source; Specimen; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Structure; Techniques; Technology; Temperature; Testing; Tissues; United States National Institutes of Health; Universities; Visible Radiation; absorption; anticancer research; antimicrobial; biological research; feasibility testing; fluorescence imaging; fluorescence microscope; fluorophore; improved; industry partner; infrared microscopy; infrared spectroscopy; insight; instrument; interest; microscopic imaging; multimodality; novel; optical imaging; protein misfolding; prototype; spectroscopic imaging; submicron; ultraviolet; vibration

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.

该第一阶段提案旨在开发并论证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分布图。 荧光标记的细胞和组织区域，然后使用以下方法对标记区域进行化学结构分析 光热红外光谱是生物研究的基石技术。 细胞和组织内目标生物分子的灵敏且高度特异性的绘图，但它不提供信息 关于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 然而，传统的红外光谱在疾病、癌症研究和其他领域有其根本的限制。 空间分辨率（即它可以分析的物体有多小）约为 10 微米，类似于一个物体的大小 因此，传统的红外光谱对于许多生物医学来说极其有限。 感兴趣的结构小于细胞大小的应用。 所提出的 FE-PTIR 技术将克服荧光显微镜和红外光谱的局限性 提供目标生物分子的高度特异性图谱以及这些分子的化学结构分析，两者 该项目将通过使用一种新颖的技术来实现这一突破。 光学光热红外光谱的形式来测量荧光标记区域的红外光谱 具体来说，FE-PTIR 技术将使用可调谐的红外激光源照射样品。 激发感兴趣的样品的分子振动将使用单独的紫外/可见光源来完成两项工作：(1) 激发样品荧光标记区域的荧光发射；以及 (2) 测量产生的局部加热； 通过测量不同区域发出的荧光强度来吸收红外辐射。 样本，可以绘制荧光标记生物分子的分布图，然后通过测量细微的变化。 由于局部红外感应加热而从样品中收集的紫外/可见光量，可以 生成相同位置和相同空间分辨率的红外吸收光谱。 然后可以使用光谱来分析样品的荧光标记区域的化学结构。 该项目与 NIH 的目标非常一致，因为它融合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、共焦显微镜和多模态 FE-PTIR 成像将非常有用，例如用荧光显微镜定位特定蛋白质，然后 使用光热红外光谱分析其结构，例如蛋白质如何折叠。 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 技术将实现一种具有亚细胞分辨率的新型多模态显微镜，该显微镜将提供 对生物医学研究具有广泛的好处，包括神经退行性疾病和抗菌素耐药性研究。

项目成果

Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level.

• DOI: 10.1021/acs.jmedchem.2c01359
• 发表时间: 2023-02-23
• 期刊: JOURNAL OF MEDICINAL CHEMISTRY
• 影响因子: 7.3
• 作者: Prater, Craig;Bai, Yeran;Konings, Sabine C.;Martinsson, Isak;Swaminathan, Vinay S.;Nordenfelt, Pontus;Gouras, Gunnar;Borondics, Ferenc;Klementieva, Oxana
• 通讯作者: Klementieva, Oxana