Advancing Deep-Tissue Multiphoton Imaging through a Novel Tunable Femtosecond Laser Source

Shih-Hsuan Chia^1*, Chi-Wen Chen¹, Hong-Yu Chou¹, Shi-Wei Chu², Li-An Chu³

¹Institute of Biophotonics, National Yang Ming Chiao Tung University, Taipei, Taiwan
²Department of Physics, National Taiwan University, Taipei, Taiwan
³Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan

* Presenter:Shih-Hsuan Chia, email:shchia@nycu.edu.tw

Ultrafast laser technology has driven significant advancements in 3D microscopy imaging, by enhancing multiphoton microscopy and enabling deep-tissue functional imaging, which is often constrained by light scattering in deeper tissues. In this work, we demonstrate that adjusting the excitation laser pulse width markedly improves the signal-to-background ratio in both two- and three-photon fluorescence imaging. Our approach employs a compact femtosecond fiber laser source with independently tunable spectral peak and bandwidth, achieved via controlled self-phase modulation. This source covers a tunable spectral range from 740-1250 nm using a Yb-based fiber laser, providing adaptable spectral coverage for diverse multiphoton bioimaging applications. Additionally, our source enables continuous spectral and bandwidth adjustment with pulse compression down to a few optical cycles.

Leveraging this bandwidth-tunable femtosecond source, we fine-tune pulse width to optimize imaging performance and introduce a new dimension for analyzing tissue scattering properties, such as effective attenuation length and anisotropy factor. Signal and background data were evaluated using beam spread functions for ballistic and scattered photons, with spatiotemporal profiles modeled after Theer and Denk’s framework. Statistical analysis of variances in multiply scattered light facilitated further tissue scattering characterization based on pulse-width-dependent signal-to-background ratios.

Our advancements in laser development and microscopy systems enable deep-tissue three-photon imaging at video frame rates. This capability has allowed functional whole-brain imaging of drosophila, showcasing the potential of this technology to capture high-resolution, real-time biological processes, thus advancing the precision and accessibility of bioimaging.

Keywords: Ultrafast optics, Nonlinear microscopy, Deep-tissue imaging, Calcium imaging