Lipid droplets have become a major research topic recently, as they are found to be involved in obesity related diseases.
Most of this research has been focused on the localization of the proteins playing a role in lipid droplet formation or
breakdown. The role of different lipid species however remains unclear because it is difficult to distinguish different fatty
acids with the present microscopy techniques. Coherent Anti-Stokes Raman scattering (CARS) is the non-linear analogue
of spontaneous Raman scattering. Multiplex CARS microscopy can provide quantitative, chemical and physical
information, making it an excellent tool to study the composition and thermodynamic phase of lipid droplets. To
investigate the potential of CARS in this field, we have incubated HeLa cells with four different fatty acids, varying in
saturation. The fatty acids were internalized by the cells and stored as lipid droplets, which we imaged with multiplex
CARS microscopy. We were able to distinguish either of the fatty acids as such in lipid droplets inside the cells.
Furthermore, we found that solid phase fatty acids were fluidized when present in lipid droplets. This illustrates the
potential of CARS microscopy to elucidate the possible role of the chemistry of fatty acids in lipid droplet regulation.
A novel procedure is developed to describe and reproduce experimental coherent anti-Stokes Raman scattering (CARS)
data, with particular emphasis on highly congested spectral regions. The approach - exemplified here with high-quality
multiplex CARS data - makes use the maximum entropy method for phase retrieval. The retrieved imaginary part of the
nonlinear susceptibility is shown to be equal to the spontaneous Raman spectrum. The phase retrieval procedure does not
influence the noise contained in the spectra. The conversion of CARS to Raman data permits a quantitative interpretation
of CARS spectra. This novel approach is demonstrated for highly congested multiplex CARS spectra of sucrose, fructose
and glucose. This novel procedures enables extraction of vibrational information from multiplex CARS data without the
use of a priori information of the sample.
A method for noninvasively determining blood oxygenation in individual vessels inside bulk tissue would provide a powerful tool for biomedical research. We explore the potential of coherent anti-Stokes Raman scattering (CARS) spectroscopy to provide this capability. Using the multiplex CARS approach, we measure the vibrational spectrum in hemoglobin solutions as a function of the oxygenation state and observe a clear dependence of the spectral shape on oxygenation. The direct extraction of the Raman line shape from the CARS data using a maximum entropy method phase retrieval algorithm enables quantitative analysis. The CARS spectra associated with intermediate oxygenation saturation levels can be accurately described by a weighted sum of the fully oxygenated and fully deoxygenated spectra. We find that the degree of oxygenation determined from the CARS data agrees well with that determined by optical absorption. As a nonlinear optical technique, CARS inherently provides the 3-D imaging capability and tolerance to scattering necessary for biomedical applications. We discuss the challenges in extending the proof of principle demonstrated to in vivo applications.
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