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

1 This Phase II project aims to develop and commercialize Fluorescence Enhanced Photothermal Infrared (FE-PTIR) imaging2 and spectroscopy. The proposed FE-PTIR will use fluorescence microscopy to map the distribution of fluorescently labeled3 regions of cells and tissue and then provide chemical structural analysis of the labeled regions using photothermal infrared4 spectroscopy. Fluorescence microscopy is a cornerstone technique in biological research, allowing sensitive mapping of5 specifically targeted biomolecules within cells and tissue, but it does not provide information about their molecular6 structure. Infrared (IR) spectroscopy can provide rich analysis of molecular structure and has been used in life sciences7 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 can9 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.