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.

Keywords: (180.5655) Raman microscopy; (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering.

Fig. 1

CARS intensity ratio (left) and phase-retrieved imaginary part of the normalized susceptibility $\Im (\overline{\tilde{\chi }})$ (right). Images (lower panels) as well as spectra (upper panels) averaged over more than 100 LDs, and for the individual droplet indicated by the yellow arrow, are shown for human adipose-derived stem cells fed with palmitic acid (PA) and α-linolenic acid (LA). Raman spectra of α-glyceryl trilinolenate (αGTL) and glyceryl trioleate (GTO) are shown for comparison (dashed lines). In the CARS intensity ratio, the spectrum of the water to glass ratio is also shown. Images of the CARS intensity ratio are shown at 1650/cm and 2850/cm, as indicated by the vertical dotted lines in the spectra. Images of the imaginary part of the normalized susceptibility are shown at 1660/cm and 2930/cm. Linear grey scales are indicated. The pump power on the sample was 20 mW (14 mW) and the Stokes power was 10 mW (7 mW) for the fingerprint region (CH region); 10 μs pixel dwell time, 0.3 μm pixel size.

Fig. 2

D-CARS imaging in the fingerprint and CH region of human ADSCs fed with different fatty acids as given in Fig. 1. Top: D-CARS spectra calculated from the measured CARS intensity ratios shown in Fig. 1 with Δ_IFD as indicated. In the CH stretch region, the inset schematically shows D-CARS amplitudes (vertical arrows) at 2920/cm and 2990/cm in relation to the CARS intensity linshape. Bottom: D-CARS images of adipocytes measured at the wavenumbers indicated by corresponding dotted lines (1470 and 1680/cm in the fingerprint region; 2920, 2990 and 3045/cm in the CH region) on a grey scale as shown. Pump power on each pair 16 mW, Stokes power on each pair 8 mW, 10 μs pixel dwell time, 0.3 μm pixel size.

Fig. 3

Ratiometric analysis of measured D-CARS ratio and phase-retrieved $\Im (\overline{\tilde{\chi }})$ in human ADSCs fed with mixtures of palmitic acid and α-linolenic acid with a vol:vol ratio as indicated. Black squares give average D-CARS intensity ratios, red triangles give average ratios of $\Im (\overline{\tilde{\chi }})$. The errors bars show the standard deviation over the analyzed droplets ensemble. Dotted lines are the calculated linear dependencies according to the mixture ratio from the values with pure PA and LA. Top: D-CARS ratio between 1680/cm and 1470/cm and corresponding ratio of $\Im (\overline{\tilde{\chi }})$ at 1660/cm and 1450/cm. Middle: D-CARS intensity ratio between 2990/cm and 2920/cm and corresponding ratio of $\Im (\overline{\tilde{\chi }})$ at 2930/cm and 2855/cm. Bottom: D-CARS intensity ratio between 3045/cm and 2855/cm and corresponding ratio of $\Im (\overline{\tilde{\chi }})$ at 3010/cm and 2855/cm.

Fig. 4

Results of FSC³ on the phase-retrived $\Im (\overline{\tilde{\chi }})$ in human ADSCs fed with PA, LA and an equal mixture (vol:vol) of PA and LA. Top: Spatial distributions of the volume concentration on a gray scale from 0 (black) to 1.1 (white) for the 5 components considered in the analysis. Bottom: spectra of $\Im (\overline{\tilde{\chi }})$ and its real part (horizontal lines) for the corresponding components. In the spectra for the PA:LA mixture the thin dotted line is an equally weighted superposition from component 4 of cells fed with PA and LA only. RGB overlays show the spatial distribution of the concentration for specific components as indicated. Below the RGB overlays, the spatial distribution of the $\Im (\overline{\tilde{\chi }})$ ratio between 2930/cm and 2855/cm is shown on a gray scale, as indicated. The scale bars indicate 20 μm.

Fig. 5

Relative spectral error E _S and relative concentration error E _C for the same cells analyzed with FSC³ in Fig. 4. The grey scale is indicated, with the range given for each image. The scale bars are 20 μm.