Demystifying Experimental Parameters in Optical Photothermal Imaging in Aqueous Media

Optical photothermal infrared (O-PTIR) spectroscopy is an indirect mid-infrared spectral imaging technique that enables label-free chemical analysis with submicrometer resolution. The technique is often used for analyzing cells and tissues and is discussed as capable of measuring in aqueous media, yet a quantitative understanding of its signal formation in liquids remains incomplete. Here, we present a systematic experimental study of transmission-mode O-PTIR supported by detailed simulations on the underlying signal generating process, focusing on O-PTIR applied to a water filled cuvette comprising two CaF₂ windows with a path length of 600 μm, by finite element analysis. We investigate the influence of moving the focal spot of the excitation mid-IR beam from the window-liquid interface into the liquid and link excitation parameters to thermal dynamics and observed readout by the probe beam. We show that the signal amplitude is governed by the pulse duration, which controls the deposited thermal energy, while the modulation frequency determines the thermal diffusion length and thus the spatial extent of the response. Depth-resolved and probe-position-dependent measurements reveal a nonlocal photothermal response dominated by refractive index gradients associated with the photothermal lens effect involving both the liquid and the windows. Time-resolved analysis further separates the temperature field into a large steady-state (DC) component and a smaller oscillatory (AC) contribution, with the latter increasing at lower modulation frequencies and both decreasing with shorter pulse durations. These results establish a unified physical picture of O-PTIR signal generation and provide a basis for practical guidelines for optimizing sensitivity, spatial resolution, and quantitative performance in liquid-phase O-PTIR measurements.