Beam scanning for rapid coherent Raman hyperspectral imaging

Coherent Raman imaging requires high-peak power laser pulses to maximize the nonlinear multiphoton signal generation, but accompanying photo-induced sample damage often poses a challenge to microscopic imaging studies. We demonstrate that beam scanning by a 3.5-kHz resonant mirror in a broadband coherent anti-Stokes Raman scattering (BCARS) imaging system can reduce photo-induced damage without compromising signal intensity. Additionally, beam scanning enables slit acquisition, in which spectra from a thin line of sample illumination are acquired in parallel during a single charge-coupled device exposure. Reflective mirrors are employed in the beam-scanning assembly to minimize chromatic aberration and temporal dispersion. The combined approach of beam scanning and slit acquisition is compared with the sample-scanning mode in terms of spatial resolution, photo-induced damage, and imaging speed at the maximum laser power below the sample-damage threshold. We show that the beam-scanning BCARS imaging method can reduce photodamage probability in biological cells and tissues, enabling faster imaging speed by using a higher excitation laser power than could be achieved without beam scanning.