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

Area of Science:

• Biomedical Optics
• Microscopy
• Laser Physics

Background:

• Coherent Anti-Stokes Raman Scattering (CARS) microscopy offers label-free chemical contrast, complementing Multiphoton Microscopy (MPM).
• Widespread adoption of multimodal CARS systems is limited by complex and untunable conventional laser sources.

Purpose of the Study:

• To develop a tunable dual-wavelength laser source for CARS microscopy.
• To integrate this source into a multimodal MPM platform for advanced imaging.

Main Methods:

• Demonstrated a tunable dual-wavelength laser source using self-phase-modulation enabled spectral selection (SESS).
• The source delivers tunable pump pulses (775-950 nm) and synchronized Stokes pulses (1.035 µm) at 37 MHz.
• Integrated the CARS source with two-photon excited fluorescence (2PEF), three-photon excited fluorescence (3PEF), second-harmonic generation (SHG), and third-harmonic generation (THG) channels.

Main Results:

• Achieved continuous Raman shift coverage from 800 cm⁻¹ to 3500 cm⁻¹ with pulse energies exceeding 2 nJ.
• Demonstrated a five-modal MPM platform: CARS, 2PEF, 3PEF, SHG, and THG.
• Successfully imaged mouse brain slices, visualizing lipids and unsaturated fatty acids with chemical specificity.

Conclusions:

• The developed tunable laser source overcomes limitations of conventional CARS systems.
• The five-modal MPM platform provides comprehensive chemical, structural, and metabolic information.
• This technology enables advanced, chemically specific imaging of biological tissues.