title: Hyperspectral and differential CARS microscopy for quantitative chemical imaging in human adipocytes.
abstract: In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200-2000/cm) and in the CH stretch region (2600-3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods.
authors.affiliation: School of Biosciences, Cardiff University, Cardiff CF10 3US, UK.
authors.forename: Claudia
authors.initials: C
authors.lastname: Di Napoli
authors.affiliation: School of Biosciences, Cardiff University, Cardiff CF10 3US, UK.
authors.forename: Iestyn
authors.initials: I
authors.lastname: Pope
authors.affiliation: School of Physics and Astronomy, Cardiff University, Cardiff CF24 3AA, UK.
authors.forename: Francesco
authors.initials: F
authors.lastname: Masia
authors.affiliation: School of Biosciences, Cardiff University, Cardiff CF10 3US, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Watson
authors.affiliation: School of Physics and Astronomy, Cardiff University, Cardiff CF24 3AA, UK.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Langbein
authors.affiliation: School of Biosciences, Cardiff University, Cardiff CF10 3US, UK.
authors.forename: Paola
authors.initials: P
authors.lastname: Borri
keywords: (180.5655) Raman microscopy; (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering