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.

这项第一阶段的提案旨在开发和论证荧光增强光热红外的可行性 (Fe-PTIR)成像和光谱分析。拟议的FE-PTIR将使用荧光显微镜来绘制 细胞和组织的荧光标记区域，然后使用 光热红外光谱。荧光显微镜是生物学研究的基石技术，允许 细胞和组织内目标生物分子的灵敏和高度特异性的图谱，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。 已被用于生命科学研究，以研究组织分类、药物/组织相互作用、神经退行性变 疾病、癌症研究等领域。然而，传统的红外光谱学对其具有基本的限制 空间分辨率(即，它可以分析的对象大致有多小)大约10微米，类似于 普通生物细胞。因此，传统的红外光谱技术在许多生物医学领域受到了极大的限制。 感兴趣的结构小于细胞大小的应用。 提出的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图谱以及这些分子的化学结构分析，两者都 与荧光显微镜具有相同的空间分辨率。这个项目将通过使用一部小说来实现这一突破 用光学光热红外光谱学的形式测量被荧光标记区域的红外光谱 样本。具体地说，FE-PTIR技术将用红外激光光源照射样品，该光源可以调节到 激发感兴趣的样本的分子振动。一个单独的紫外线/可见光光源将用于两项工作：(1)至 在样品的荧光标记区域激发荧光发射；以及(2)测量产生的局部加热 来自红外辐射的吸收。通过测量从不同区域发出的荧光的强度， 样品，就有可能绘制荧光标记的生物分子的分布。然后通过测量 从样品中收集的由于局部红外感应加热而产生的UV/可见光的量，可以 产生相同位置和相同空间分辨率的红外吸收光谱。红外线吸收 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。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推动力 和生物工程，包括光学成像和光谱、红外成像、共聚焦显微镜和多模式 成像。Fe-PTIR将非常有用，例如在荧光显微镜下定位特定蛋白质，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 折叠错误是许多神经退行性疾病(如阿尔茨海默氏症)的根本原因，FE-PTIR将提供新的见解。 展示FE-PTIR技术将使一种新的具有亚细胞分辨率的多模显微镜能够提供 为包括神经退行性疾病和抗菌素耐药性研究在内的生物医学研究带来深远的好处。

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