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Cellular lipid droplets are organelles that carry out important functions such as the regulated storage and release of cholesterol and fatty acids (FAs). Lipid droplets therefore play a key role in both healthy metabolism and metabolic disorders such as obesity, type 11 diabetes, and atherosclerosis. Hence, there has been much interest in understanding the chemical (lipid) composition and physical state (lipid packing) of lipid droplets. Coherent anti-Stokes Raman Scattering (CARS) microscopy has proven to be a useful tool in lipid droplet research because of its ability to visualize lipid droplets in a label-free manner. Multiplex CARS microscopy in spectroscopy mode has been particularly useful: In conjunction with appropriate spectral processing, it allows one to obtain the local Raman response with submicron spatial resolution. Thus, multiplex CARS microscopy provides rapid quantitative information and allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual lipid droplets. We discuss the different vibrational markers that may be used to characterize the lipid droplet properties and demonstrate that while the C-H stretch of unsaturated =C-H groups is a useful marker of the level of acyl unsaturation in vitro, it fails in vivo. Moreover, we present here results on two different cell lines - adipocytes and HeLa cells - with distinct lipid droplet characteristics, both in terms of lipid droplet size and dependence of the lipid droplet composition on the incubation medium. Copyright (C) 2009 John Wiley & Sons, Ltd.

Erratum to ‘‘Ultrafast vibrational dynamics of interfacial water” [Chem. Phys. 350 (2008) 23] Avishek Ghosh , Marc Smits , Maria Sovago , Jens Bredenbeck , Michiel Muller , Mischa Bonn a,* a FOM-Institute for Atomic and Molecular Physics (AMOLF), Kruislaan 407, NL-1098 SJ, Amsterdam, The Netherlands b Swammerdam Institute for Life Sciences, University of Amsterdam, P.O. Box 94062, 1090 GB Amsterdam, The Netherlands

In situ quantitative imaging of concentration profiles of reactants and products inside a microfluidic reactor is achieved, with submicron spatial resolution with mM sensitivity and on ms time scales, for a given position. The label-free approach relies on quantitative vibrational spectroscopy, using Coherent Anti-Stokes Raman scattering microscopy in a spectrally resolved fashion, and is demonstrated on an elementary acid-base reaction.

A Reply to the Comment by C. S. Tian and Y. R. Shen.Received 26 August 2008DOI:https://doi.org/10.1103/PhysRevLett.101.139402©2008 American Physical Society

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.

Nonlinear optical effects can be successfully exploited to generate a quantitative metric of molecular, organelle, cellular, and indeed organ-level dynamics. Further, nonlinear optics can be used to create the very tools essential for these dynamic nonlinear optical studies. We will present our recent results that use nonlinear optics to fabricate novel optical devices which, in turn, are used for nonlinear spectroscopy and imaging.

We report a femtosecond time-resolved study of water at the neat water-air interface. The O-H stretch vibrational lifetime of hydrogen-bonded interfacial water is measured using surface-specific 4th-order nonlinear optical spectroscopy with femtosecond infrared pulses. The vibrational lifetime in the frequency range of 3200 to 3500 cm(-1) is found to closely resemble that of bulk water, indicating ultrafast exchange of vibrational energy between surface water molecules and those in the bulk.

The effect of sodium and calcium ions on zwitterionic and anionic phospholipids monolayers is investigated using vibrational sum-frequency generation in conjunction with surface pressure measurements and fluorescence microscopy. Sodium ions only subtly affect the monolayer structure, while the effect of calcium is large and depends strongly on the surface pressure. At low surface pressures (approximately 5 mN/m), the presence on Ca2+ results in the unexpected appearance of ordered domains. For pressures between approximately 5 and approximately 25 mN/m, Ca2+ ions induce disorder in the monolayer. For pressures exceeding 25 mN/m, calcium cations expand the monolayer, while simultaneously ordering the lipid chains. Interestingly, effects are similar for both zwitterionic lipids and negatively charged lipids. In both vibrational sum-frequency generation and surface tension measurements, the molecular signature of the association of Ca2+ with the lipids is evident from Ca2+-induced changes in the signals corresponding to area changes of 4 A2/lipid-precisely the surface area of a Ca2+ ion, with evidence for a change in lipid Ca2+ complexation at high pressures.

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.

We report on the energy flow dynamics in model membranes, investigated by surface-specific time-resolved (femtosecond) sum frequency generation spectroscopy. This recently developed technique allows us to probe energy dynamics selectively at the water/lipid interface. We report vibrational relaxation dynamics of C-H stretch modes in the lipid alkyl chains, and reveal that incoherent energy transfer occurs from the excited CH2 groups to the terminal CH3 groups. We also find evidence for strong anharmonic coupling between different CH2 and CH3 modes. Relaxation and the energy transfer processes within the lipid alkyl chain occur on (sub-)picosecond timescales. Studies of the dynamics on different lipid phases (gel or liquid crystalline phase) reveal a marked independence of the dynamics on the precise molecular conformation of the lipids. In addition, we report the energy transfer dynamics between membrane- bound water and lipids, and find that the transfer of heat between water and lipids occurs remarkably fast: heat is transferred across the monolayer, from the polar head group region of the lipid to the end of the alkyl chain, within 1 ps. These results demonstrate the potential of using ultrafast surface-specific spectroscopies to elucidate biomolecular dynamics at membrane surfaces.

Coherent anti-Stokes Raman scattering microscopy develops rapidly into a powerful technique to image both the chemical composition and physical state in complex samples from biophysics, biology, and the material sciences. This nonlinear vibrational technique increases the signal relative to spontaneous Raman scattering and does not require labeling of the specimen. A theoretical description of the technique is provided and the two major modes of operation: picosecond- and multiplex coherent anti-Stokes Raman scattering are discussed. The potential of the technique is demonstrated with examples of direct measurement of acyl chain order and orientation in lipid monolayers, bilayers, and lipid vesicles.

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 implementation of broad-bandwidth sum-frequency generation (SFG) spectroscopy is presented, which allows for the simultaneous recording of SFG spectra with different polarization combinations of the SFG, visible, and infrared beams. This method is particularly advantageous for studies in which surface properties are time-dependent, such as kinetic studies. The technique is illustrated by a study that mimics lung surfactant relaxation during the breathing cycle. The time-dependent molecular order of lung surfactant lipids is quantified through the vibrational response of the terminal CH3 group.

The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid–cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (ld), solid-ordered (so) and liquid-ordered (lo) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at ∼27 and ∼22 mol% for ld–lo and so–lo coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule.

Coherent anti-Stokes Roman scattering (CARS) microscopy is presented as a new nonlinear optical technique. The combination of vibrational spectroscopy and microscopy allows highly sensitive investigations of unlabelled samples. CARS is an ideal tool for studying a broad variety of samples. The main drawback of the technique is its non-zero-background nature, which implies that the signal has to be detected against a nonresonant background. The need to solve this problem is reflected in the rapid technological developments that have been observed during the last decade. Recent results show that CARS microscopy has the potential to become an important complementary technique that can be used with other well-established microscopic methods. Although it has some limitations, it offers unique access to many problems that cannot be tackled with conventional techniques. For this reason, it can be expected that the impressive growth of the field will continue.

Third Harmonic Generation (THG) from the vicinity of interfaces, using focused laser beams can be obtained virtually from any inhomogeneous medium. Its sensitivity to the presence and extent of inhomogeneity in the focal volume has already found a variety of applications ranging from material characterization to label free three-dimensional microscopy of biological samples. In this presentation, we demonstrate a number of new applications of THG in the microscopy of food samples and living cells. Also, we report on an anomalous behavior in the THG z-response. So far the observations and theoretical predictions supported a single peak of THG signal, with the peak position corresponding to the interface. We have observed an anomalous behavior where a single interface can give rise to two peaks located across the interface. The simulations, which we carried out using a paraxial theory of THG and measurements done on typical normally dispersive materials, suggest that this anomalous behavior is due to a particular combination of χ(3) and the magnitude of dispersion.