Mid-infrared (mid-IR) spectroscopy and optoacoustic/optothermal (OA/OT) imaging are perfectly complementary technologies to each other. Vibrational molecular excitations by mid-IR absorption are utterly de-excited in the form of heat while efficient OA/OT signal generation primarily depends on heat deposition. This synergy allows overcoming the (otherwise) persistent limitations of traditional mid-IR spectroscopy and imaging in live-cell/fresh-tissue applications— i.e., sample opacity due to water absorption. Combination of mid-IR excitation and OA/OT detection has resulted in new tools for label-free live-cell, tissues, and in vivo metabolic research. Here we discuss basic principles on mid-IR detection for spectroscopy and imaging as well as the most recent developments on mid-IR OA and OT microscopy that overcome the limitations of conventional vibrational spectroscopy for biosensing and label-free metabolic microscopy.
Imaging modalities based on vibrational spectroscopy (Raman or mid-IR imaging) have demonstrated high label-free chemical specificity for different biomolecules. Nevertheless, conventional mid-IR microscopy have been limited mostly to dry tissues and fixed cells due to the strong mid-IR absorption of water and due to the use of conventional negative-contrast detection. We introduce positive-contrast Mid-infraRed Optoacoustic Microscopy (MiROM) for label-free metabolic imaging in living cells. We showcase the unique capabilities of MiROM in living cells by monitoring the spatiotemporal distribution of carbohydrates, lipids, and proteins in adipocytes during lipogenesis as well as monitoring the lipid-protein dynamics in brown and white adipocytes during lipolysis. We discuss how MiROM yields unique label-free metabolic imaging abilities for a broader range of bioanalytical studies in living cells and its potential for analytical histology in unprocessed tissues.
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