Evidence is accumulating that in cell membranes microdomains exist, also referred to as rafts or detergent resistant membranes. In this study, atomic force microscopy is used to study supported lipid bilayers, consisting of a fluid phosphatidylcholine, sphingomyelin and cholesterol. Domains were visualized of which the morphology and size depended on the cholesterol concentration. The presence of cholesterol was found to induce bilayer coupling. At 30 mol% cholesterol, a change in percolation phase was observed, and at 50 mol%, when both fluid lipids and solid lipids are saturated with cholesterol, phase separation was still observed. In addition, we were able to directly visualize the resistance of domains against non-ionic detergent. ß

Direct extraction of Raman line-shapes from congested CARS spectra.Vartiainen, E.M.; Rinia, H.A.; Müller, M.; Bonn, M. Published in: Optics Express DOI:10.1364/OE.14.003622 Link to publication Citation for published version (APA):Vartiainen, E. M., Rinia, H. A., Müller, M., & Bonn, M. (2006). Direct extraction of Raman line-shapes from congested CARS spectra. Optics Express, 14(8), 3622-3630. DOI: 10.1364/OE.14.003622 General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Abstract:We show that Raman line-shapes can be extracted directly from congested coherent anti-Stokes Raman scattering (CARS) spectra, by using a numerical method to retrieve the phase-information hidden in measured CARS spectra. The proposed method utilizes the maximum entropy (ME) model to fit the CARS spectra and to further extract the imaginary part of the Raman susceptibility providing the Raman line-shape similar to the spontaneous Raman scattering spectrum. It circumvents the challenges arising with experimentally determining the real and imaginary parts of the susceptibility independently. Another important advantage of this method is that no a priori information regarding the vibrational resonances is required in the analysis. This permits, for the first time, the quantitative analysis of CARS spectra and microscopy images without any knowledge of e.g. sample composition or Raman response. ©2006 Optical Society of AmericaOCIS codes: (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering; (000.3860) General, mathematical methods in physics.References and links

Lipid droplets (LDs) are highly dynamic organelles that perform multiple functions, including the regulated storage and release of cholesterol and fatty acids. Information on the molecular composition of individual LDs within their cellular context is crucial in understanding the diverse biological functions of LDs, as well as their involvement in the development of metabolic disorders such as obesity, type II diabetes, and atherosclerosis. Although ensembles of LDs isolated from cells and tissues were analyzed in great detail, quantitative information on the heterogeneity in lipid composition of individual droplets, and possible variations within single lipid droplets, is lacking. Therefore, we used a label-free quantitative method to image lipids within LDs in 3T3-L1 cells. The method combines submicron spatial resolution in three dimensions, using label-free coherent anti-Stokes Raman scattering microscopy, with quantitative analysis based on the maximum entropy method. Our method allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual LDs. Our results reveal variations in lipid composition and physical state between LDs contained in the same cell, and even within a single LD.

Expression of phosphatidylserine (PtdSer) at the cell surface is part of the membrane dynamics of apoptosis. Expressed phosphatidylserine functions as an "eat me" flag toward phagocytes. Here, we report that the expressed phosphatidylserine forms part of a hitherto undescribed pinocytic pathway. Annexin A5, a phosphatidylserine-binding protein, binds to and polymerizes through protein-protein interactions on membrane patches expressing phosphatidylserine. The two-dimensional protein network of annexin A5 at the surface prevents apoptotic body formation without interfering with the progression of apoptosis as demonstrated by activation of caspase-3, PtdSer exposure, and DNA fragmentation. The annexin A5 protein network bends the membrane patch nanomechanically into the cell and elicits budding, endocytic vesicle formation, and cytoskeleton-dependent trafficking of the endocytic vesicle. Annexin A1, which binds to PtdSer without forming a two-dimensional protein network, does not induce the formation of endocytic vesicles. This novel pinocytic pathway differs from macropinocytosis, which is preceded by membrane ruffling and actin polymerization. We clearly showed that actin polymerization is not involved in budding and endocytic vesicle formation but is required for intracellular trafficking. The phosphatidylserine-annexin A5-mediated pinocytic pathway is not restricted to cells in apoptosis. We demonstrated that living tumor cells can take up substances through this novel portal of cell entry. This opens new avenues for targeted drug delivery and cell entry.

The maximum entropy method for phase retrieval of multiplex coherent anti-Stokes Raman scattering (CARS) spectra is described in detail and applied to the time-resolved measurement of the main lipid phase transition of small, unilamellar 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles subject to a 3 min temperature sweep. Since the--thus derived--imaginary part of the third-order CARS susceptibility can be directly related to the linear vibrational spectrum, the multiplex CARS spectral data can be analyzed quantitatively and without prior knowledge of the sample. It is shown that the maximum entropy model provides an exact description of the original data, including the noise, if all available autocorrelation functions are used. Our findings confirm the acyl-chain order-disorder phase-transition behavior of small, unilamellar lipid vesicles.