Ludwig-Soret microscopy with the vibrational photothermal effect.

Vibrational microscopy provides label-free, bond-selective chemical contrast by detecting molecular vibrations, making it invaluable for biomedical research. While conventional methods rely on the direct detection of Raman scattering or infrared absorption, recently developed vibrational photothermal (ViP) microscopy achieves chemical contrast indirectly through refractive index (RI) changes. This indirect approach enables unique imaging capabilities beyond traditional chemical imaging. Here, we introduce an application of ViP microscopy: Label-free intracellular thermophoretic (Soret) imaging, which visualizes biomolecular transport driven by temperature gradients. ViP-induced Soret (ViPS) imaging leverages a steady-state temperature distribution generated by optical heating through vibrational photothermal effect, combined with time-resolved RI imaging via optical diffraction tomography. Using ViPS imaging, we measured thermophoretic behavior in living COS7 cells, determining intracellular diffusion and Soret coefficients. Notably, we observed a reversed direction of molecular transport (negative Soret effect) in the cytoplasm compared to the nucleus, possibly driven by thermophoresis-induced diffusiophoresis. Furthermore, time-lapse imaging under CO₂-depleted conditions revealed a remarkable reduction in thermophoretic activity, suggesting glass formation during the dying process, likely due to polymer aggregation. ViPS imaging represents a frontier in intracellular thermophoretic studies, expanding the capabilities of vibrational microscopy.