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