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.
Highlights
Optical microscopy is an indispensable tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution within living cells and tissues
The coherent anti-Stokes Raman scattering (CARS) intensity ratio has a spectral shape affected by the interference between the resonant vibrational contribution and the non-resonant electronic contribution to the CARS susceptibility resulting in a dispersive lineshape in the fingerprint region and a spectral shape only qualitatively resembling a Raman spectrum in the CH stretch region
In this work we have compared the performances of fast dual-frequency CARS imaging, requiring only minimal data analysis, with hyperspectral CARS imaging analyzed with phaseretrieval and an unsupervised factorization into concentrations of chemical components (FSC3), for quantitative chemical imaging on human stem-cell derived adipocytes fed with saturated and poly-unsaturated fatty acids
Summary
Optical microscopy is an indispensable tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Coherent anti-Stokes Raman scattering microscopy has emerged in the last decade as a multiphoton technique able to image living cells label-free in real time with high threedimensional spatial resolution and chemical specificity [1, 2]. The recent development of CARS micro-spectroscopy has further boosted this interest by opening the prospect to image LDs in living cells with an unprecedented intrinsic chemical specificity and without introducing staining artifacts. Such artifacts include for example cytosolic LD fusion artificially induced by standard staining protocols [5,6,7]. It became possible to gain detailed quantitative insights about the fusion behavior of unstained LDs in adipocytes with CARS time-course experiments over more than a week on the same cells without the limitation of marker bleaching [8]
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