Here we proposed an imaging strategy to monitor more fluorescent objectives synchronously in the living animals with multicolor two-photon excited fluorescence microscopy. We used highly nonlinear photonic crystal fibers and a 100-fs Ti: Sapphire oscillator to generate 200 nm wide continuum pulses, which covers the two-photon excitation spectra of conventional fluorophores. Then, we used the phase shaping method to compensate for the dispersion and switch excitation wavelength rapidly. Next, we implemented non-negative matrix factorization (NMF) to unmix images with cross-talk. Images of 8-color HeLa cells and 4-color mice tumor under the mouse dorsal skinfold chamber were acquired.
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