Two-photon excited fluorescence (TPEF) microscopy provides spectacular insights into visualization of cellular events within intravital tissue due to advantages of inherent sectioning ability, relatively deep optical penetration, and low optical damage. However, because the excitation spectrum of multiple fluorophores (FPs) are often separated, the spectral band of standard 100-fs Ti: Sapphire oscillators is not sufficiently board to efficiently excite multiple FPs. By pumping pulses from the 100-fs Ti: Sapphire oscillator through the highly nonlinear photonic crystal fibers (HNPCF), continuum pulses with 700-900 nm spectra covering common FPs can be generated due to enhanced self-phase modulation. We demonstrated that broadband fiber continuum combined with phase shaping technology can be ideal for multicolor TPEF microscopy. By phase shaping the continuum pulses, rapid selective excitation of specified FPs can be achieved to decrease the spectral crosstalk of multiple FPs in multicolor TPEF microscopy. We achieved selective excitation which allows in vivo imaging of four FPs in skin fold window chamber of mouse. The result shows that the selective excitation efficiency and contrast ratios are both high. Phase shaping selective excitation using fiber continuum is expected to optimally excite and separate multiple FPs when synchronously monitoring the dynamic characteristics of a variety of cells in tumor microenvironment. This work shows potential to promote multicolor two-photon fluorescence microscopy in the application for in vivo imaging.
Quan Cui, Ling Fu, Xinyuan Huang, Zhongyun Chen, and Min Chen, "Multicolor selective excitation two-photon microscopy by phase-shaping with broadband fiber-continuum (Conference Presentation)," Proc. SPIE 10493, Dynamics and Fluctuations in Biomedical Photonics XV, 104930T (Presented at SPIE BiOS: January 29, 2018; Published: 15 March 2018); https://doi.org/10.1117/12.2289008.5752208513001.
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