Hilde A. Rinia

Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy

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.

Direct extraction of Raman line-shapes from congested CARS spectra.

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.

Imaging orientational order and lipid density in multilamellar vesicles with multiplex CARS microscopy

Multiplex coherent anti‐Stokes Raman scattering (CARS) microscopy is used to measure the width of the orientational order distribution of lipid acyl chains within a three‐dimensionally confined microscopic probing volume. A theoretical model is developed to describe and simulate the polarization‐dependent measurements. We observe that the orientational order in phosphatidylcholine multilammellar vesicles increases significantly upon addition of small amounts (≤ 15 mol%) of cholesterol and is significantly reduced for unsaturated lipids. Based on these measurements and using the quantitative nature of multiplex CARS microscopy the exact local concentration of lipid molecules within the vesicles can be measured in terms of the number of lipid bilayers present in the microscopic probing volume.

Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering

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.

Chapter 4 Visualization and Characterization of Domains in Supported Model Membranes

Abstract Lateral segregation of lipid and protein components within biomembranes can lead to the formation of biologically functional domains. This chapter reviews microdomain formation in supported model membranes, induced by lipid–lipid interactions. The discussed techniques used for bilayer imaging are atomic force microscopy and fluorescence microscopy. Furthermore, we describe the promising capabilities and thus far obtained results of multiplex coherent anti-stokes Raman scattering microscopy with respect to functional imaging of microdomains in lipid model systems.

Quantitative multiplex CARS spectroscopy in congested spectral regions

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.

Multiplex coherent anti-Stokes Raman scattering microscopy on lipid droplets in HeLa cells

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.