Highly Nonlinear Dynamics of In Vivo Deep-Tissue Interaction with Femtosecond Laser Pulses at 1030 nm

Abstract We report on the highly nonlinear behavior observed in the central nervous system tissue of zebrafish (Danio rerio) when exposed to femtosecond pulses at 1030 nm. At this irradiation wavelength, photo damage becomes detectable only after exceeding a specific peak intensity threshold, which is independent of the photon flux and irradiation time, distinguishing it from irradiation at shorter wavelengths. Furthermore, we investigate and quantify the role of excessive heat in reducing the damage threshold, particularly during high-repetition-rate operations, which are desirable for label-free and multi-dimensional microscopy techniques. To verify our findings, we examined cellular responses to tissue damage, including apoptosis and the recruitment of macrophages and fibroblasts at different time points post-irradiation. These findings substantially contribute to advancing the emerging nonlinear optical microscopy techniques and provide a strategy for inducing deep-tissue, precise and localized injuries using near-infrared femtosecond laser pulses.