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Deep Tissue Multiphoton Microscopy Based on Advanced Femtosecond Fiber Sources

The use of multiphoton microscopy has proven its potential on deep tissue virtual biopsy for early diagnosis and neuronal processes imaging. However, the capability of multiphoton microscopy strongly depends on the availability of easily operated femtosecond laser sources. By precise management of fiber-optic nonlinearity, we have demonstrated the way to generate nearly transform-limited femtosecond sources with the tunable wavelength range of 0.9 µm – 1.2 µm, covering both the GFP two-photon-excitation band and transparency window of common bio-tissues. The demonstrated approach is fully energy scalable to sub-100 nJ level of pulse energy with the spectral tunable range down to 1.31 µm, which paves the way to capture deep-tissue imaging contrast from third harmonic generation and three-photon-excited GFP fluorescence. We applied such an energetic source to a proof-of-principle study of ex vivo human skin based on harmonics (i.e., second-harmonic generation and third-harmonic generation) imaging. This new type of fiber-format energetic ultrafast source will be performed as a robust solution for multiphoton microscopy applications, as well as studying the damage assessments from thermal heating and multiphoton absorption during the imaging process.