In vivo and simultaneous multimodal imaging: Integrated multiplex coherent anti-Stokes Raman scattering and two-photon microscopy

Simultaneous multimodal imaging is critical for in vivo study of biological dynamic processes and clinical applications. In this study, we demonstrate a multimodal nonlinear optical (NLO) microscopy of the capability to simultaneously produce images of multiplex coherent anti-Stokes Raman scattering (M-CARS), two-photon excitation fluorescence, and second harmonic generation in living biological systems. The excitation sources are a femtosecond Ti:sapphire laser and a broadband supercontinuum generated from a photonic crystal fiber. The simultaneously excited multiple NLO signals were recorded by using a time- and wavelength-resolved detection technique. In M-CARS imaging, the nonresonant background is effectively reduced by using a simple background subtraction method.