Ultrahigh-resolution and single-molecule stimulated Raman scattering (SRS) microscopy

Project summary Super-resolution optical microscopy promises to revolutionize biological imaging, as it enables non-invasive interrogation at molecular scale. Indeed, the emergence of super-resolution fluorescence microscopyhas quickly impacted the way biologists study cells and subcellular phenomenon. However, super-resolution fluorescence microscopy has fundamental limitations due to the use thefluorescence as contrast mechanism. In particular, it has three major limitations: (1) it cannot reveal chemicalcomposition of the sample; (2) it cannot interrogate small biomolecules due to the relatively bulky fluorescenttags; (3) it cannot image a large number of targets due to the color barrier (only 2~5 fluorescent colors can bepractically resolved).The goal of this project is to develop a novel super-resolution imaging platform by exploiting stimulatedRaman scattering (SRS) as the contrast mechanism. During the past 10 years since its invention in 2008, SRSmicroscopy has made widespread impact in biomedical imaging. Being a chemically sensitive method, SRS iswell known for its label-free chemical analysis in a quantitative manner. With the recent development of tinybio-orthogonal tags such as alkynes, SRS has been proven successful in interrogating a wide spectrum ofsmall biomolecules such as lipids, glucose, amino acids, and drugs. Very recently, novel vibrational dyepalettes with fine spectral resolution have been reported to achieve super-multiplex electronic pre-resonance(epr) SRS imaging of more than 20 targets simultaneously. Importantly, all these utilizes of SRS microscopy islimited by light diffraction. With SRS being a perfectly complementary contrast mechanism to the prevalent fluorescence, thecurrent proposal aims to develop the necessary methods to bring SRS microscopy to the realm of superresolution. (1) How to improve the resolution for general chemical imaging and small biomolecule imaging; (2)how to break the diffraction limit of the super-multiplex epr-SRS imaging; (3) how to develop the matchingvibrational dyes for single molecule SRS. Towards these goals, we had laid out a systematic plan as to how to crystallize this concept into apowerful technology platform. An inter-disciplinary approach has been planned out including instrumentationdevelopment, computational imaging, and novel probes synthesis. In Aim 1, we will develop and build newinstrumentation. In Aim 2, we will explore new computational algorithm. In Aim 3, we will design next-generation vibrational probes. If successfully implemented, we will establish a transformative platform. Theresulting super-resolution chemical imaging would find wide applications in systematically unraveling complexbiological systems such as neuroscience, immunology and cancer biology for basic research, diseasediagnostics and precision medicine.1