The ability to observe samples qualitatively at the microscopic scale has greatly enhanced our understanding of the physical and biological world throughout the 400 year history of microscopic imaging, but there are relatively few techniques that can truly claim the ability to quantify the local concentration and composition of a sample. We review coherent anti-Stokes Raman scattering (CARS) as a quantitative, chemically specific, and label-free microscopy. We discuss the complicating influence of the nonresonant response on the CARS signal and the various experimental and mathematical approaches that can be adopted to extract quantitative information from CARS. We also review the uses to which CARS has been employed as a quantitative microscopy to solve challenges in material and biological science.

The digestion and absorption of lipophilic, bioactive molecules such as lipids, physiologically active nutrients (nutraceuticals), and drugs play a crucial role in human development and health. These molecules are often delivered in lipid droplets. Currently, the kinetics of digestion of these lipid droplets is followed by in vitro models that simulate gastrointestinal conditions, while phase changes within the lipid droplets are observed by light or electron microscopy. However real-time, spatially resolved information about the local chemical composition and phase behavior inside the oil droplet is not accessible from these approaches. This information is essential as the surface and phase behavior determine the local distribution of molecules in the oil droplets and thus may influence the rate of uptake, for example, by impairing the effective transfer of bioactive molecules to intestinal cells. We demonstrate the capability of multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to image the digestion process non-invasively, with submicrometer resolution, millimolar sensitivity, and without the need for labeling. The lipolysis of glyceryl trioleate emulsion droplets by porcine pancreatic lipase is imaged, and the undigested oil and the crystalline lipolytic products are distinguished by their different vibrational signatures. The digestion of droplets containing the phytosterol analogue ergosterol is also probed, and the crystals are observed to dissolve into the lipolytic products. The lipophilic drug progesterone and Vitamin D(3) are dissolved in glyceryl trioctanoate emulsion droplets, and the local concentration is mapped with millimolar sensitivity. The bioactive molecules are observed to concentrate within the droplets as the oil is hydrolyzed. This observation is ascribed to the low solubility of these molecules in the lipolytic products for this system. Neither the type of bioactive molecule nor the initial radius of the emulsion droplet had a large effect upon the rate of digestion under these conditions; lipolysis of the triglyceride by pancreatic lipase appears insensitive to the type of bioactive molecule in solution. These findings shed important new light on lipid digestion and open new possibilities for the chemical visualization of lipid digestion and phase changes in lipid droplets containing bioactive molecules, which in combination with other existing techniques will provide a full picture of this complex physicochemical process.

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The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.