Instantaneous Phase-Shifting Optothermal Microscopy for Live-cell Metabolic Monitoring

Cellular metabolic activities can be studied using label-free vibrational spectroscopic imaging to leverage the endogenous contrast of biomolecules. However, fast live-cell imaging over large fields-of-view remains challenging due to the need for raster scanning and despite advances in wide-field modalities, imaging rapid cellular activities across large cell populations remains challenging. Here, we introduce a mid-infrared optothermal microscopy method termed Instantaneous Phase-Shifting Optothermal Microscopy (IPSOM). IPSOM circumvents conventional mechanical phase-shifting methods, achieved single-frame imaging speed 588-fold increase compared to the sequential approach, under field-of-view of 300×350 µm. For multi-wavelength hyperspectral imaging, IPSOM achieves an 8-fold speed improvement. IPSOM is used here to monitor lipid remodelling in adipocytes during lipolysis, demonstrating its potential for studying rapid cellular metabolism.