Multiplexed Brain and Visceral Two-Photon Imaging Using a Simulation-Guided Ultrafast Three-Color Fiber Laser

Multicolor two-photon microscopy is an essential tool in modern life sciences, enabling simultaneous, high-resolution imaging of multiple cellular structures and dynamic processes within complex biomedical systems. Realizing its full potential demands light sources that combine multiplexed spectral flexibility, high pulse quality, and practical implementation for efficient excitation of diverse cellular targets. Here, we present a novel ultrafast fiber laser platform that enables efficient three-color multiplexed two-photon imaging through numerically optimized nonlinear spectral shaping in a photonic crystal fiber (PCF). The system is driven by a nonlinear Yb-doped fiber amplifier with tailored dispersion and gain characteristics to generate clean sub-50 fs pulses at 1030 nm with over 40 nJ pulse energy. Subsequent, simulation-guided PCF-based spectral broadening enables controlled formation of three distinct high-energy bands centered at 940 nm, 1,080 nm, and 1,175 nm, overlapping with key fluorescent probes and biomolecular markers. The resulting pulses, isolated with high spectral and time-domain pulse quality, provide sub-115 fs duration and 2.5 - 6 nJ energy per channel. Multiplexed imaging is validated in labeled mouse brain, kidney, and liver tissue slices using spectrally independent multi-fluorophore targeting to visualize e.g., astrocytes, neuronal structures, and nuclei in triple-stained mouse hippocampus. The demonstrated fiber-optic laser platform provides a practical alternative to conventional single-color sources and more complex multi-laser systems, supporting robust and high-resolution three-color two-photon imaging for a range of biomedical applications.