Multiphoton Microscope with a Rapid Broadband CARS Modality

Abstract

Multiphoton microscopy utilizing pulsed near-infrared (NIR) laser pulses has facilitated many developments in the biological and materials sciences. Multiphoton excitation fluorescence (MPEF), most notably two-photon excitation fluorescence (2PEF), allows high-resolution 3D sectioning with greater penetration depth in tissues due to NIR excitation [1]. Second harmonic generation (SHG) microscopy allows sensitive imaging of materials with non-centrosymmetric inversion symmetries (e.g. collagen) [1], and surface third-harmonic generation (THG) microscopy provides contrast at interfaces with strong heterogeneity in optical properties [2]. Nonlinear Raman techniques such as coherent anti-Stokes Raman spectroscopy (CARS) provide chemically-specific contrast related to the vibrational structure of the sample [3]. While CARS imaging with picosecond excitation and spectral detection is typically limited to a narrow spectral range, Fourier-transform (FT-) CARS techniques using femtosecond excitation provide broadband Raman capabilities [4–8].