Deterministic Fiber‐Optic Spectral Engineering Enables Three‐Color Multiplexed Two‐Photon Microscopy

ABSTRACT Multicolor two‐photon microscopy is a powerful tool for simultaneous, high‐resolution imaging of multiple cellular structures and dynamics in complex biological systems. However, its broader adoption remains limited by the complexity of existing excitation light sources, which typically rely on multi‐laser architectures, cascaded parametric conversion schemes, or heuristic broadband fiber sources. Here, we present a deterministic, simulation‐guided spectral‐engineering framework, realized in a compact ultrafast fiber‐laser platform, that enables precise design of efficient and balanced three‐color two‐photon excitation. By systematically designing and numerically co‐optimizing a dispersion‐ and gain‐engineered Yb‐doped fiber laser with subsequent nonlinear spectral shaping in a photonic crystal fiber (PCF), we achieve controlled formation of three energetic and spectrally isolated excitation bands centered at 960, 1080, and 1175 nm. Each band delivers 2.5–5.8 nJ pulse energy with sub‐115 fs duration, well‐matched to the two‐photon excitation bands of widely used fluorescent probes, without the need for multiple laser sources or parametric conversion stages. Multiplexed two‐photon imaging of triple‐stained mouse brain, kidney, and liver tissues demonstrates robust, spectrally independent visualization of fine structures, including astrocytes, neuronal architectures, and nuclei. This work establishes deterministic nonlinear spectral engineering in fiber optics as a predictive and practical route to compact, wavelength‐flexible excitation sources for high‐performance multicolor two‐photon microscopy.