Unveiling Hidden Signatures in Life by Vibrational Photothermal Microscopy

Project Summary Optical microscopy is a fundamental tool for life science. Phase contrast microscope allows visualization of intracellular structures. Fluorescence microscope further allows real-time imaging of tagged molecules or organelles. Yet, the fluorescent tags such as fluorescent proteins are larger than many molecules inside a cell and their broad spectral profile prohibits highly multiplexed imaging. A central theme of the Cheng Research Group is to fill this gap in biomolecular imaging through invention and development of advanced bond-selective microscopy tools. Over the past 5 years, under the support of a MIRA grant R35GM136223, Cheng and co- workers have made substantial progress in development and applications of stimulated Raman scattering (SRS) microscopy and mid-infrared photothermal (MIP) microscopy, reported in 42 peer-reviewed articles and 16 patent applications. Despite these advances, there remains a need to develop highly sensitive vibrational imaging technologies and demonstrate killer applications to life science and/or medical research. While providing micromolar imaging sensitivity, the MIP technology is not sensitive to Raman-active bonds such as C-H or alkyne. Either SRS or MIP microscopy does not allow for high-resolution vibrational imaging of biomolecules in deep tissue environment. Thus far, most vibrational imaging applications are focused on high-concentration molecules such as lipid droplets or protein aggregates in cultured cells or thin tissue slices. Broader applications to address challenges not addressable by fluorescence imaging are needed to prove the value of vibrational imaging. This MIRA renewal is designed to address these needs in the next 5 years. First, Cheng and co-workers will develop stimulated Raman photothermal (SRP) microscopy to enable ultrasensitive Raman spectroscopic imaging. Based on a pilot demonstration (Science Advances, 2023), PI and co-workers will further build an OPA-based SRP microscope to reach micro-molar imaging sensitivity and apply this technique to image cholesterol-rich domains in live cells. PI and co-workers will also build a fiber-OPO based SRP microscope for broad use of this technology at ease of operation. Second, PI and co-workers will develop short-wave infrared photothermal (SWIP) microscopy to enable deep-tissue vibrational imaging with millimeter- scale penetration depth and sub-micron spatial resolution. Based on a pilot demonstration (Nature Photonics, 2024), PI and co-workers will further develop a SWIP-OCT system to enable high-speed volumetric bond- selective imaging of tumor spheroids, organoids, and deep tissues in their natural states. Finally, PI and coworkers will demonstrate a few paradigm-shifting applications of vibrational photothermal microscopy via collaboration with scientists in life science. Applications will include fingerprinting intracellular organelles through fluorescence-detected MIP microscopy, click-free imaging of transport and metabolism of small biomolecules, developing and imaging vibrational proteins to study protein machinery in live cells and in vivo, and MIP reporter (Nature Methods 2024) based super-multiplex spatial omics.