The endogenous nonlinear optical (NLO) signals of two-photon excitation fluorescence (TPEF), second harmonic
generation (SHG), and coherent anti-stokes Raman scattering (CARS) have been widely used to image a variety of
biological samples. Different nonlinear optical signals could convey different structural and biomolecular information.
Therefore, it is desirable to combine multiple nonlinear optical signals together for biomedical imaging. However, the
simplification of the sophistical, high cost laser source and the simultaneous excitation and detection of multiple NLO
signals are the challenges for the multimodal NLO microscopy. In this work, we instrument a multimodal nonlinear
optical microscopy system which integrates the multiplex CARS module with the TPEF, SHG microscopy. The
excitation source is the combination of femtosecond Ti:sapphire laser and the broadband near infrared supercontinuum
light from a photonic crystal fiber. The multiplex CARS measurements, covering the vibrational frequency from 2400 to
3300 cm-1, allowed us to detect the pure non-resonant background (NRB) signals and the CARS signals of aliphatic C-H
and O-H bonds simultaneously. The relatively large NRB in the femtosecond laser excited CARS images could be
efficiently suppressed by simple subtraction operation. The TCSPC detection system records the spectral and temporal
characteristics of the TPEF signals and spectrally resolves the CARS signals from different molecular vibrational bonds.
We demonstrate the multimodal imaging capability of the system using C.elegans as the living biological samples.