Integrated coherent anti-Stokes Raman scattering and multiphoton microscopy for biomolecular imaging using spectral filtering of a femtosecond laser

Abstract

We report on a unique implementation of a dual 4-f paired-gratings spectral filtering of a femtosecond (fs) laser to realize high contrast coherent anti-Stokes Raman scattering (CARS) microscopy and high quality multiphoton microscopy on the same platform for label-free biomolecular imaging. The 4-f gratings filtering enables readily conversion of the 100 fs laser pulse trains into transform-limited 1 picosecond (ps) pulses in the spectral range of 700-1600 nm for CARS imaging. Compared with fs-CARS microscopy, our ps-CARS imaging yields an approximately tenfold improvement in spectral resolution, while a twofold improvement in nonresonant background suppression. High quality second-harmonic generation and two-photon excitation fluorescence imaging can also be acquired in tandem when swapping the 4-f grating filtering from the ps mode to fs mode. We demonstrate this multimodal nonlinear optical microscopy technique by imaging 465 nm polystyrene beads in water and animal liver tissue. © 2010 American Institute of Physics.