Simultaneous label-free autofluorescence-multiharmonic microscopy driven by femtosecond sources based on self-phase modulation enabled spectral selection

Nonlinear optical microscopy techniques have unique advantages in tissue imaging, such as enhanced contrast, high resolution, label-free deep optical sectioning capabilities and so on. Nonlinear optical microscopy also has multiple imaging modalities, corresponding to various components in biological tissues. Unfortunately, its wide applications are hindered due to the lack of broadly tunable femtosecond sources designed for driving multimodalities simultaneously. To solve this challenge, we proposed a new wavelength conversion approach——self-phase modulation (SPM) enabled spectral selection, dubbed as SESS. SESS employs SPM to broaden the input spectrum in a short fiber, and the broadened spectrum features well-isolated spectral lobes. Using suitable optical filters to select the outermost spectral lobes produces nearly transform-limited femtosecond pulses. In this paper, we demonstrate a fiber-optic SESS source for multimodal nonlinear optical microscopy. Based on a 43-MHz Yb-fiber laser, this SESS source can emit 990-nm, 84-fs pulses with 5-nJ energy and 84-fs pulse duration; it can also produce 1110-nm, 48-fs pulses with 15-nJ energy. The 990-nm pulses are used to drive two-photon excitation fluorescence of many important fluorophores and second-harmonic generation microscopy, which, combined with image splicing technology, enables us to obtain large field of view image of the gastric tissue. We also employ the 1110-nm pulses to drive simultaneous label-free autofluorescence-multiharmonic microscopy for multimodal imaging of gastric tissue. Two-photon excitation fluorescence, three-photon excitation fluorescence, second-harmonic generation and third-harmonic generation signals of gastric tissue are simultaneously excited efficiently. Such a multimodal nonlinear optical microscopy driven by SESS sources constitutes a powerful tool for driving multimodal nonlinear optical microscopy in biomedical imaging.