Project Summary A genuine label-free imaging technology, vibrational microscopy provides maps of cells and tissues with exceptionally high chemical contrast as it directly probes the fundamental vibrational modes of samples. Vibrational imaging approaches include IR-absorption micro-spectroscopy and confocal Raman microscopy, methods that have been successfully commercialized (a growing 500 million dollar market) and are now common tools of inquiry found in analytical and biological laboratories. Over the past four decades, these techniques have had a measurable impact in the fields of biology and biomedicine, offering a spatially resolved assessment of healthy and diseased tissues from a molecular perspective. This proposal aims to significantly improve the capabilities of vibrational microscopy. We propose a new imaging approach that merges the desirable properties of IR absorption microscopy with some of the unique properties of coherent, nonlinear optical (NLO) excitation of molecules. This novel IR-NLO technique improves the spatial resolution of IR absorption microscopy by tenfold, while offering higher sensitivity to fingerprint molecular vibrations relative to Raman-based microscopy methods. Our team is comprised of experts in coherent Raman scattering microscopy and IR microspectroscopic imaging. Our innovation makes it possible to rapidly acquire IR absorption images of fingerprint vibrational modes with a resolution of 0.5 micrometer or better. The preliminary data shows that the IR-NLO approach can be successfully adopted in a rapid laser-scanning microscope, allowing convenient vibrational imaging of tissue specimens. In our proposal we develop, test, and improve the new IR-NLO technology. The validation of the technology is achieved through extensive biomedical imaging studies and comparison with the state of the art IR microscopy available today. The proposed program tackles a major challenge in IR spectroscopic microscopy, namely the improvement of imaging resolution. This new capability is significant, as the higher resolution enables the identification of sub-micrometer intra- and extra-cellular structures in the tissue, which hitherto have remained invisible in IR-imaging. The high-resolution imaging property thus dramatically improves the diagnostic capabilities of the technique. By setting a new resolution standard for fingerprint vibrational imaging, the IR-NLO technology is likely to have a significant impact in tissue imaging and can enable its use in both research and clinical domains for pathology.