Five-modal multiphoton microscopy enabled by a tunable dual-wavelength fiber laser

Coherent anti-Stokes Raman scattering (CARS) microscopy provides chemically specific, label-free contrast that complements the structural and functional information offered by multiphoton microscopy (MPM). However, the widespread adoption of multimodal CARS systems has been hindered by the complexity and limited tunability of conventional solid-state laser sources. We demonstrate a tunable dual-wavelength laser source based on self-phase-modulation enabled spectral selection (SESS), delivering pump pulses tunable from 775 nm to 950 nm together with synchronized Stokes pulses at 1.035 µm. The source operates at a repetition rate of 37 MHz with pulse energies exceeding 2 nJ and enables continuous Raman shift coverage from 800 cm −1 to 3500 cm −1 . By integrating this CARS excitation source with multiphoton excitation channels, we demonstrate a five-modal MPM platform; that is, CARS, two-photon excited fluorescence (2PEF), three-photon excited fluorescence (3PEF), second-harmonic generation (SHG), and third-harmonic generation (THG). Multimodal imaging of mouse brain slices demonstrates chemically selective visualization of lipids and unsaturated fatty acids, together with complementary structural and metabolic contrasts.