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

Area of Science:

• Biophotonics
• Microscopy
• Cellular Imaging

Background:

• Live-cell microscopy requires minimal sample perturbation for accurate observation.
• Light-sheet microscopy enhances sample health by confining excitation to a single plane.
• Laser-scanning nonlinear optical microscopy, despite restricted excitation, faces challenges in gentle, label-free molecular imaging.

Purpose of the Study:

• To investigate the mechanism of phototoxicity in laser-scanning nonlinear optical microscopy.
• To identify strategies for mitigating phototoxicity in label-free molecular imaging.
• To enable gentle, real-time imaging of biological samples.

Main Methods:

• Developed a phototoxicity assay using time-lapse auto-fluorescence elevation in a chicken breast tissue model.
• Investigated phototoxicity induced by intense near-infrared excitation in laser-scanning microscopy.
• Proposed and validated a rapid scanning technique with sub-80-fs excitation and full triplet relaxation.

Main Results:

• Provided strong evidence that linear absorption, not nonlinear absorption, is the primary cause of phototoxicity.
• Demonstrated that rapid scanning with full triplet relaxation significantly mitigates phototoxicity.
• Achieved label-free imaging of freely moving *C. elegans* in real-time.

Conclusions:

• Linear absorption of intrinsic biomolecules leading to triplet buildup is the dominant phototoxicity mechanism.
• A simple, rapid scanning technique effectively reduces phototoxicity across various sample types.
• This approach facilitates gentle, label-free live imaging with high irradiance tolerance.