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

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.