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 extremel...