Gentle Label‐Free Nonlinear Optical Imaging Relaxes Linear‐Absorption‐Mediated Triplet

Abstract Sample health is critical for live‐cell fluorescence microscopy and has promoted light‐sheet microscopy that restricts its ultraviolet–visible excitation to one plane inside a 3D sample. It is thus intriguing that laser‐scanning nonlinear optical microscopy, which similarly restricts its near‐infrared excitation, has not broadly enabled gentle label‐free molecular imaging. It is hypothesized that intense near‐infrared excitation induces phototoxicity via linear absorption of intrinsic biomolecules with subsequent triplet buildup, rather than the commonly assumed mechanism of nonlinear absorption. Using a reproducible phototoxicity assay based on the time‐lapse elevation of autofluorescence (hyper‐fluorescence) from a homogeneous tissue model (chicken breast), strong evidence is provided supporting this hypothesis. The study justifies a simple imaging technique, e.g., rapidly scanned sub‐80‐fs excitation with full triplet‐relaxation, to mitigate this ubiquitous linear‐absorption‐mediated phototoxicity independent of sample types. The corresponding label‐free imaging can track freely moving C. elegans in real‐time at an irradiance up to one‐half of water optical breakdown.