Thermal lensing during infrared neural stimulation enables spatially resolved photothermal dosimetry

Photothermal laser tissue interactions (LTI) are challenging to study at the subcellular level due to the complexity of accurately characterizing spatial energy distributions. Infrared neural stimulation (INS), a label-free photothermal neuromodulation technique using pulsed infrared light, has demonstrated promise but lacks standardized, high-resolution dosimetry methods. In this study, we present an automated, imaging-based workflow to perform spatially resolved photothermal dosimetry. This method uses thermal lensing to mark the location of IR exposure within the imaging field of view, enabling precise assessment of the radiant exposure dosage and correlated neuronal responses. Neuronal calcium responses to single infrared pulses of varying duration (350 µs, 2 ms, and 8 ms) were measured using widefield fluorescence microscopy. The thermal lensing artifact (TLA) observed during stimulation was used to model the spatial energy distribution of the laser beam profile. Neuronal Ca²⁺ responses were analyzed relative to the local radiant exposure, H₀(x,y), and the average radiant exposure, dosage, Havg, calculated using the laser pulse energy divided by the laser spot area. The TLA provided a reliable fiducial for tracking the IR stimulus within the imaging field. Neuronal responses to INS were spatially dependent and exhibited three phenotypes: unreactive, low-amplitude, and high-amplitude. The Gaussian laser beam profile led to cells near the beam center receiving higher radiant exposure dosages, exceeding activation thresholds. We find that shorter pulse durations required lower radiant exposure dosages to elicit neuronal responses. The Havg consistently underestimates the radiant exposure required for stimulation. The H₀(x,y) required for stimulation did not produce measurable cellular damage. Local radiant exposure dosage dictates neuronal activation during INS. Our method provides a standardized, high-throughput approach for performing spatially resolved photothermal dosimetry at microscopic level.