A Femtosecond Fiber Laser-Based Compact Label-Free Multimodality Nonlinear Optical Microscopy and Its Ex Vivo Bioimaging

The study introduces a novel multimode nonlinear optical microscopy system utilizing an Er3+-doped femtosecond fiber laser, capable of performing second harmonic generation (SHG), third harmonic generation (THG), and three-photon fluorescence (3PEF) imaging simultaneously. This system successfully visualized various biological structures, including plant cell walls, starch particles, chloroplasts, onion cell linkers, and skin structures such as the stratum corneum, epidermal intercellular structures, hair follicles, and collagen. The spatial resolution achieved was 1.67 μm for 3PEF, 1.5 μm for THG, and 2.67 μm for SHG. The improved spatial resolution with a high N.A objective allows for diverse biological applications, including in vivo label-free imaging and deep microscopic imaging.