Video-rate volumetric chemical imaging via mid-infrared photothermal optical diffraction tomography

Abstract

Label-free vibrational microscopy provides chemically specific access to cellular structure, yet quantitative volumetric chemical dynamics in living cells remain largely inaccessible, particularly on subsecond timescales relevant to intracellular transport and structural reorganization. This limitation arises because most high-speed vibrational techniques rely on raster scanning, which constrains volumetric throughput to approximately one volume per second (vps). Although mid-infrared photothermal (MIP) imaging offers a pathway toward spatially parallel chemical detection, existing implementations have remained far below video-rate volumetric operation, reflecting a fundamental trade-off between imaging speed and signal-to-noise ratio. Here, we overcome this trade-off in MIP tomography and realize video-rate volumetric chemical imaging using mid-infrared photothermal optical diffraction tomography (MIP-ODT), achieving high photothermal sensitivity while maintaining quantitative measurement fidelity. High per-angle detectability supports volumetric reconstruction without temporal averaging, yielding a signal-to-noise ratio exceeding 70 under video-rate acquisition conditions. Consequently, volumetric imaging at 19.2 vps is achieved, representing a nearly 400-fold improvement over prior implementations. Using this capability, we performed video-rate three-dimensional tracking of lipid droplets in living cells and quantified anomalous diffusion from full volumetric trajectories, revealing heterogeneous intracellular transport behaviors that are obscured in two-dimensional measurements. We further demonstrate high-speed hyperspectral volumetric chemical imaging across a 300 cm-1 spectral window within 1 s through rapid MIR wavenumber sweeping, paving the way for real-time three-dimensional organelle-specific chemical phenotyping.