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

Nonlinear multimode imaging is a versatile tool to realize complex structural and compositional information of biological samples. In this study, we presented a novel integrated multimode nonlinear optical microscopy system by using an Er3 + -doped femtosecond fiber laser. The system could perform second harmonic generation (SHG), third harmonic generation (THG), and three-photon fluorescence (3PEF) imaging modes simultaneously. Using this imaging system, we detected signals from plant cell walls, as well as starch particles in potatoes, chloroplasts in leaves, and onion cells wall-to-membrane linkers: Hechtian strands. Visualizations of skin structures such as stratum corneum, epidermal intercellular structures, hair follicles, and collagen were also achieved by a spatial resolution of 1.67 μm for 3PEF, 1.5 μm for THG, and 2.67 μm for SHG modalities, respectively. After spatial resolution improved by changing a high N.A objective, the system could be used for a variety of biological applications, including in vivo label-free imaging of living animals and deep microscopic imaging.