Submicron Infrared Imaging of Molecular Vibrations through Third-Order Upconversion

Infrared (IR) spectroscopy offers direct access to molecular vibrational modes, making it a cornerstone technique in biomedical research, chemical analysis, and materials science. Its inherently large absorption cross-section and low photon energy enable label-free imaging with minimal phototoxicity─an ideal platform for studying living systems. However, conventional IR microscopy remains fundamentally constrained by long wavelengths, resulting in limited spatial resolution and reduced detection sensitivity. Here, we introduce mid-infrared third-order sum-frequency generation (ITS) imaging, a new spectroscopic modality that transforms vibrational absorption signatures in the mid-IR into visible-frequency signals through a third-order nonlinear process. ITS circumvents the longstanding challenges in IR microscopy by enabling diffraction-limited visible-light resolution and efficient detection with silicon photodetectors. By extracting absorption spectra from the ITS signals, our method enables the reconstruction of chemically specific spectral features for polymers, proteins, and lipids. To establish its biological relevance, we map the distributions of proteins and lipids in living Haematococcus pluvialis cells through spectral reconstruction using multivariate curve resolution, achieving a 0.46 μm spatial resolution and subpicosecond temporal precision. This label-free and biocompatible approach provides both chemically specific contrast and high imaging throughput, opening the door to real-time visualization of dynamic biochemical processes in living systems.