Increasing the imaging depth of coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.