1420
J. Phys. Chem. B 2008, 112, 1420-1426
Three-Dimensional Molecular Mapping of a Multiple Emulsion by Means of CARS Microscopy Tobias Meyer,† Denis Akimov,‡ Nicolae Tarcea,‡ Susana Chatzipapadopoulos,† Gerald Muschiolik,§ Jens Kobow,‡ Michael Schmitt,‡ and Ju1 rgen Popp*,†,‡ Institute of Photonic Technology, Jena, Germany, Institute of Physical Chemistry, Friedrich-Schiller-UniVersita¨t Jena, Germany, and Institute of Nutrition, Friedrich-Schiller-UniVersita¨t Jena, Germany ReceiVed: October 2, 2007; In Final Form: NoVember 12, 2007
Multiple emulsions consisting of water droplets dispersed in an oil phase containing emulsifier which is emulsified in an outer water phase (W/O/W) are of great interest in pharmacology for developing new drugs, in the nutrition sciences for designing functional food, and in biology as model systems for cell organelles such as liposomes. In the food industry multiple emulsions with high sugar content in the aqueous phase can be used for the production of sweets, because the high sugar content prevents deterioration. However, for these emulsions the refractive indexes of oil and aqueous phase are very similar. This seriously impedes the analysis of these emulsions, e.g., for process monitoring, because microscopic techniques based on transmission or reflection do not provide sufficient contrast. We have characterized the inner dispersed phase of concentrated W/O/W emulsions with the same refractive index of the three phases by micro Raman spectroscopy and investigated the composition and molecular distribution in water-oil-water emulsions by means of threedimensional laser scanning CARS (coherent anti-Stokes Raman scattering) microscopy. CARS microscopy has been used to study water droplets dispersed in oil droplets at different Raman resonances to visualize different molecular species. Water droplets with a diameter of about 700 nm could clearly be visualized. The advantages of CARS microscopy for studying this particular system are emphasized by comparing this microscopic technique with conventional confocal reflection and transmission microscopies.
Introduction Emulsions are of great importance for everyday life. Foods like milk and many kinds of cosmetics (e.g., beauty creams) are emulsions. They are widely used in the nutrition sciences and in pharmacology. Therefore a rapid technique for the analysis of the composition of these mixtures is desirable, for instance, for process monitoring. Multiple emulsions, which consist of three phases (W/O/W or O/W/O) separated by membrane-like interfaces, are very promising for designing drugs and functional foods, because delicate ingredients within the inner aqueous phase are protected from destructive influences such as oxidation by the surrounding oil layer. Furthermore, these emulsions can be especially designed for releasing ingredients of the inner watery phase slower, for instance in functional food at a certain point of digestion. Beyond that multiple emulsions are interesting systems because they are similar in composition to cell organelles like liposomes. Hence fundamental diffusion processes can be studied with the help of this artificial system. In the food industry these emulsions can be used in sweets. However, to protect the mixture from deterioration, the sugar content of the aqueous phase needs to be in the range of 65%. For this sugar content the refractive indexes of the watery and the oil phase become very similar; hence conventional microscopic techniques based on transmission and reflection do not provide sufficient contrast for visualizing the composition of * Corresponding author. E-mail:
[email protected]. † Institute of Photonic Technology. ‡ Institute of Physical Chemistry, Friedrich-Schiller-Universita ¨ t Jena. § Institute of Nutrition, Friedrich-Schiller-Universita ¨ t Jena.
the emulsion and for displaying the distribution of its main components. There exist different microscopic methods that are based on miscellaneous contrast mechanisms which allow visualizing the spatial distribution of the refractive index gradient or phase differences, fluorescing species, and specific molecular bonds. While most microscopic techniques are based on imaging differences in transmission or fluorescence of the various components, they cannot be applied to an emulsion lacking any change in refractive index or which does not contain fluorescing species. Furthermore, the molecular specificity of these techniques is rather low and therefore they cannot be used to determine the composition of the sample directly. Staining the sample, on the other hand, can change the properties of the emulsion; hence this technique is not always applicable. In this work we demonstrate three-dimensional imaging of multiple emulsion droplets using coherent anti-Stokes Raman scattering (CARS) microscopy. This vibrational imaging technique can directly visualize the molecular composition of a complex sample, if several characteristic Raman resonances are used. Since the frequency of a molecular vibration is dependent on the mass of the atoms involved and the strength of the bonds between them, it is highly specific for a molecule. Especially vibrations within the fingerprint spectral region (
