Label-free multimodal imaging with simultaneous two-photon and three-photon microscopy and kernel-based nonlinear...

Area of Science:

• Biomedical Optics
• Microscopy
• In Vivo Imaging

Background:

• Simultaneous multimodal imaging offers enhanced biological tissue characterization.
• Label-free imaging techniques are crucial for minimizing perturbation of biological samples.
• Existing multimodal systems often face limitations in simultaneous acquisition and signal separation.

Purpose of the Study:

• To develop a compact system for simultaneous two-photon microscopy (2PM) and three-photon microscopy (3PM).
• To acquire multiple label-free contrasts including two-photon-excitation-fluorescence (2PEF), second harmonic generation (SHG), and third harmonic generation (THG).
• To address challenges in dual-wavelength excitation, signal separation, and data processing for multimodal imaging.

Main Methods:

• Development of a compact multimodal imaging system with dual excitation wavelengths.
• Simultaneous acquisition of 2PM and 3PM signals.
• Implementation of a kernel-based nonlinear scaling (KNS) denoising method for ultra-low signal images.
• Demonstration on various biological tissue samples.

Main Results:

• Successful simultaneous acquisition of 2PM and 3PM images with multiple label-free contrasts (2PEF, SHG, THG).
• Effective separation of 2PM and 3PM signals and management of varying signal levels.
• High-quality multimodal images generated using the KNS denoising method.
• Demonstration of reduced motion artifacts and mechanical drift compared to sequential acquisition.

Conclusions:

• The developed system enables simultaneous multimodal imaging with 2PM and 3PM.
• The KNS method effectively denoises ultra-low signal images, improving image quality.
• Simultaneous acquisition accelerates imaging and minimizes motion artifacts, showing great potential for label-free in vivo imaging of biological tissues.