Multimodal nonlinear optical imaging has opened new opportunities and becomes a powerful tool for imaging complex
tissue samples with inherent 3D spatial resolution.. We present a robust and easy-to-operate approach to add the coherent
anti-stokes Raman scattering (CARS) imaging modality to a widely used multiphoton microscope. The laser source
composed of a Mai Tai femtosecond laser and an optical parametric oscillator (OPO) offers one-beam, two-beam and
three-beam modalities. The Mai Tai output at 790 nm is split into two beams, with 80% of the power being used to pump
the OPO. The idler output at 2036 nm from OPO is doubled using a periodically poled lithium niobate (PPLN) crystal.
This frequency-doubled idler beam at 1018 nm is sent through a delay line and collinearly combined with the other Mai
Tai beam for CARS imaging on a laser-scanning microscope. This Mai Tai beam is also used for multiphoton
fluorescence and second harmonic generation (SHG) imaging. The signal output at 1290 nm from OPO is used for SHG
and third-harmonic generation (THG) imaging. External detectors are installed for both forward and backward detection,
whereas two internal lamda-scan detectors are employed for microspectroscopy analysis. This new system allows
vibrationally resonant CARS imaging of lipid bodies, SHG imaging of collagen fibers, and multiphoton fluorescence
analysis in fresh tissues. As a preliminary application, the effect of diacylglycerol acyltransferase 1 (DGAT1) deficiency
on liver lipid metabolism in mice was investigated.