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

Area of Science:

• Biophotonics
• Microscopy
• Cellular Imaging

Background:

• Live-cell microscopy demands gentle sample preparation to maintain cellular health.
• Light-sheet microscopy uses planar excitation for reduced photodamage.
• Laser-scanning nonlinear optical microscopy (LS-NLM) has not achieved broad adoption for gentle, label-free molecular imaging despite restricted excitation.

Purpose of the Study:

• To investigate the mechanism of phototoxicity in laser-scanning nonlinear optical microscopy.
• To propose and validate a method for mitigating phototoxicity in label-free imaging.
• To enable gentle, real-time molecular imaging of live biological samples.

Main Methods:

• Developed a phototoxicity assay using time-lapse autofluorescence elevation (hyper-fluorescence) in a chicken breast tissue model.
• Investigated phototoxicity induced by intense near-infrared excitation in laser-scanning nonlinear optical microscopy.
• Implemented a rapid scanning technique with sub-80-femtosecond excitation and full triplet relaxation.

Main Results:

• Provided strong evidence that phototoxicity arises from linear absorption by intrinsic biomolecules and subsequent triplet buildup, not nonlinear absorption.
• Demonstrated that the proposed rapid scanning technique effectively mitigates linear-absorption-mediated phototoxicity across different sample types.
• Achieved real-time, label-free imaging of freely moving Caenorhabditis elegans (C. elegans) at high irradiance levels.

Conclusions:

• Linear absorption is the primary driver of phototoxicity in laser-scanning nonlinear optical microscopy.
• A simple, rapid scanning imaging technique can overcome phototoxicity limitations.
• This approach facilitates gentle, label-free molecular imaging for live biological samples, including C. elegans.