Hyperspectral volumetric coherent anti-Stokes Raman scattering microscopy: quantitative volume determination and NaCl as non-resonant standard

Abstract

In this work, we demonstrate quantitative volume determination of chemical components in three dimensions using hyperspectral coherent anti-Stokes Raman scattering microscopy, phase-corrected Kramers-Kronig retrieval of the coherent anti-Stokes Raman scattering susceptibility and factorization into concentration of chemical components. We investigate the influence of the refractive index contrast between water and polymer beads (polystyrene and polymethylmethacrylate), showing that it leads mainly to concentration errors, while the spectral error is less affected. The volume of polystyrene beads of sizes from 200 nm to 3 μm is determined with 10% relative error and 1% absolute error in the region of interest. We furthermore establish the use of sodium chloride as non-resonant reference material free of Raman-active vibrational resonances.

Keywords: CARS microscopy; hyperspectral image analysis.