Temporally-optimized spectrally-shaped hyperspectral coherent anti-Stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy offers label-free chemical contrasts based on molecular vibrations. Hyperspectral CARS (HS-CARS) microscopy enables comprehensive microscale chemical characterization of biological samples. Various HS-CARS methods have been developed with individual advantages and disadvantages. We present a new temporally-optimized and spectrally-shaped (TOSS) HS-CARS method to overcome the limitations of existing techniques by providing precise control of the spatial and temporal profiles of the excitation beams for efficient and accurate measurements. This method uniquely uses Fourier transform pulse shaping based on a two-dimensional spatial light modulator to control the phase and amplitude of the excitation beams. TOSS-HS-CARS achieves fast, stable, and flexible acquisition, minimizes photodamage, and is highly adaptable to a multimodal multiphoton imaging system.