Gentle label-free nonlinear optical imaging relaxes linear-absorption-mediated triplet

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 three-dimensional 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. We hypothesize 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 auto-fluorescence (hyper-fluorescence) from a homogeneous tissue model (chicken breast), we provide strong evidence supporting this hypothesis. Our 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.