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bioRxiv · 10.1101/2023.06.17.545432

Phase-shifting mid-infrared optothermal microscopy for wide-field hyperspectral imaging of living cells

Yuan, T.·Riobo, L.·Gasparin, F.·Ntziachristos, V.·Pleitez, M. A.
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Abstract

Fast live-cell hyperspectral imaging at large field-of-views (FOVs) and high cell confluency remains challenging in vibrational microscopy due to the need for point-by-point focal excitation scanning. Imaging at high cell confluency and large FOVs is important, respectively, for proper cell function and statistical significance of measurements. Here, we introduce phase-shifting mid-infrared optothermal microscopy (PSOM) which interprets molecular-vibrational information as the optical path difference (OPD) induced by mid-infrared absorption and is capable of taking snapshot vibrational images over broad mid-infrared excitation areas at high live-cell confluency. By means of phase-shifting, PSOM suppresses noise to a quarter of current optothermal microscopy modalities to allow capturing live-cell vibrational images at FOVs up to 50 times larger than state-of-the-art. Additionally, it reduces illumination power flux density (PFD) down to 5 orders of magnitude lower than conventional vibrational microscopy--thus, considerably decreasing the possibility of cell photodamage.

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Yuan, T., Riobo, L., Gasparin, F., Ntziachristos, V., Pleitez, M. A.. 2023-06-18. Phase-shifting mid-infrared optothermal microscopy for wide-field hyperspectral imaging of living cells. https://doi.org/10.1101/2023.06.17.545432

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