Optical-trapping Raman microscopy detection of single unilamellar lipid vesicles

Raman spectra of individual unilamellar phospholipid vesicles ( approximately 0.6 microm in size) have been acquired by optical-trapping confocal Raman microscopy over the 900-3200-cm(-)(1) region. Raman scattering from the phospholipid bilayer of a single, trapped liposome could be detected, along with molecular species trapped within the vesicle. The Raman spectra of vesicles prepared from four different phosphatidylcholine lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), could be readily distinguished by evaluating differences in the skeletal C-C and C-H stretching modes of the acyl hydrocarbon tails. These differences correlate with changes in lipid organization for different gel to liquid-crystal transition temperatures (T(m)): 41, 24, 7, and -20 degrees C for DPPC, DMPC, DLPC, and DOPC, respectively. The spectra could be acquired on the same trapped vesicle for several hours, which allowed the permeability of the bilayer to be investigated by monitoring the leakage of perchlorate anions from the vesicle. Vesicles prepared from pure DPPC or DOPC, with gel to liquid-crystal transition temperatures well above and well below room temperature, exhibited no detectable anion transfer. DLPC and DMPC vesicles permitted rapid ion transfer across the bilayer. The lengths of hydrocarbon tails were shorter in these two lipids, which could indicate that shorter chains lower the hydrophobic barrier of a membrane to ion transport. While the DMPC chains were longer than DLPC with a correspondingly higher T(m), the temperature of the experiment corresponds to the T(m) of DMPC, and domain boundaries between gel and liquid-crystal phases could contribute to high membrane permeability.     

Optically trapping confocal Raman microscopy of individual lipid vesicles: kinetics of phospholipase A(2)-catalyzed hydrolysis of phospholipids in the membrane bilayer

Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 microm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C-C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s(-1). Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied.     

Detection of drug-membrane interactions in individual phospholipid vesicles by confocal Raman microscopy

Optical-trapping confocal Raman microscopy is developed as a method to study the interactions of drugs or other compounds with the membranes of individual phospholipid vesicles. This technique allows membrane disorder, permeability, and drug localization to be assessed without the need for labeling of the membrane or the compounds of interest. We have applied this technique to study the interactions of two nonsteroidal antiinflammatory drugs, salicylate and ibuprofen, with vesicles prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The results show that both salicylate and ibuprofen increase membrane disorder, as determined from increases in the Raman scattering from gauche conformers in the phospholipid acyl chains. By monitoring the Raman scattering of the drug molecules in optically trapped DMPC vesicles, the membrane permeability and partitioning of the drugs could be determined; the spatial distributions of the drugs were also measured by scanning the laser focus through surface-adhered 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles, producing a profile of the vesicle and its contents. Though the membrane is permeable to both drugs, ibuprofen preferentially accumulates in the membrane, whereas salicylate does not. The measured ibuprofen accumulation agrees quantitatively with the water/octanol partition coefficient of the drug and the estimated volume of the lipid membrane. The results suggest that ibuprofen localizes in the hydrophobic acyl chain region of the membrane, whereas salicylate weakly associates with the phospholipid headgroups.     

Raman spectroscopic analysis of biochemical changes in individual triglyceride-rich lipoproteins in the pre- and postprandial state

Individual triglyceride-rich lipoprotein (TGRL) particles derived from human volunteers are nondestructively analyzed by laser tweezers Raman microspectroscopy, and information on their composition and distribution is obtained. The Raman signature of single optically trapped very low-density lipoproteins (VLDL), a subclass of TGRL, which play an important role in cardiovascular disease, exhibits distinct peaks associated with molecular vibrations of fatty acids, proteins, lipids, and structural rearrangements of lipids. Our analysis of pre- and postprandial VLDL exhibits the signature of biochemical changes in individual lipoprotein particles following the consumption of meals. Interaction of VLDL with endothelium leads to the breakdown of complex triacylglycerols and the formation of a highly ordered core of free saturated fatty acids in the particle. A particle distribution analysis reveals trends in the degree to which this process has occurred in particles at different times during the postprandial period. Differences in particle distributions based on the different ratios of polyunsaturated to saturated fats in the consumed meals are also easily discerned. Individual lipoprotein particles hydrolyzed in vitro through addition of lipoprotein lipase (LpL) exhibit strikingly similar changes in their Raman spectra. These results demonstrate the feasibility of monitoring the dynamics of lipid metabolism of individual TGRL particles as they interact with LpL in the endothelial cell wall using Raman spectroscopy.     

Probing chirality of a lipid tubular by confocal Raman microscopy

The chiral phospholipids 1,2-bis-(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9 PC) can self assemble into lipid nanotubules. This hollow cylindrical supramolecular structure shows promise in a number of biotechnological applications. The mechanism of lipid tubule formation was initiated by assembling of lipid bilayer sheets from amphiphilic solution. Upon cooling, small ribbons were detached from the sheets and rolled up into helical tubules. The lipid tubules obtained were 0.6-0.8 microm in diameter and approximately 50 microm in length. Raman spectra of individual polymerized lipid tubules were measured by focused laser excitation of 532 nm leading to intense and reproducible Raman spectra. The chirality of lipid tubules was investigated by atomic force microscopy (AFM) and confocal Raman microscopy. We report the Raman mapping images revealing helical tubular profiles of C=C stretching and C[triple bond]C stretching of lipid tubules. Circular dichroism property of lipid tubules has also been probed with a 532 nm laser.     

Spatially resolved analysis of small particles by confocal Raman microscopy: depth profiling and optical trapping

Raman microscopy is a powerful method to provide spatially resolved information about the chemical composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles, either resting on a surface or optically trapped at a laser focus, where the confocal collection volume optimizes the signal from the particle. In this work, the sensitivity and spatial selectivity of detecting Raman scattering from single particles was determined as a function of particle size. An inverted confocal Raman microscope was used to acquire spectra of individual surface-bound and optically trapped polystyrene particles with sizes ranging between 200 nm and 10 microm. The particles are in contact with aqueous solution containing perchlorate ion that served as a solution-phase Raman-active probe to detect interferences from the surrounding medium. The collection volume is scanned through single particles that are attached to the surface of the coverslip, and the sensitivity and selectivity of detection are measured versus particle size. The results compare favorably with a theoretical analysis of the excitation profile and confocal collection efficiency integrated over the volumes of the spherical particles and the surrounding solution. This analysis was also applied to the detection of particles that are optically trapped and levitated above the surface of the coverslip. The results are consistent with the optical trapping of particles at or near the excitation beam focus, which optimizes excitation and selective collection of Raman scattering from the particle.     

Discriminating and imaging different phosphatidylcholine species within phase-separated model membranes by principal component analysis of TOF-secondary ion mass spectrometry images

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) enables chemically imaging the distributions of various lipid species in model membranes. However, discriminating the TOF-SIMS data of structurally similar lipids is very difficult because the high intensity, low mass fragment ions needed to achieve submicrometer lateral resolution are common to multiple lipid species. Here, we demonstrate that principal component analysis (PCA) can discriminate the TOF-SIMS spectra of four unlabeled saturated phosphatidylcholine species, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) according to variations in the intensities of their low mass fragment ions (m/z ≤ 200). PCA of TOF-SIMS images of phase-separated DSPC/DLPC and DPPC/DLPC membranes enabled visualizing the distributions of each phosphatidylcholine species with higher contrast and specificity than that of individual TOF-SIMS ion images. Comparison of the principal component (PC) scores images to atomic force microscopy (AFM) images acquired at the same membrane location before TOF-SIMS analysis confirmed that the PC scores images reveal the phase-separated membrane domains. The lipid composition within these domains was identified by projection of their TOF-SIMS spectra onto PC models developed using pure lipid standards. This approach may enable the identification and chemical imaging of structurally similar lipid species within more complex membranes.     

Buckling of lipid tubules in shrinking liquid droplets

Self-assembled hollow lipid tubules are interesting and potentially useful supramolecular structures. Here, we study the deformation of lipid tubules of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) trapped inside liquid droplets on glass substrates. The interface tension of the shrinking liquid droplets exerts a compression force on the ends of the trapped lipid tubules, and causes them to buckle. This provides a method to measure their mechanical properties. The Young's modulus of the DC8,9PC lipid tubules is estimated to approximately 1.07 GPa. As the strain energy of the buckled tubules builds up, they poke through the interface of shrinking liquid droplets and then adhere onto glass substrates to form looplike shapes.     

Ultrasonic trapping of microparticles in suspension and reaction monitoring using Raman microspectroscopy

An ultrasonic standing wave around 2 MHz has been used for trapping and concentration of suspended micrometer-size particles in a flow cell, whereas Raman microspectroscopy was used as a nondestructive technique to provide molecular information about the trapped particles. With this approach, detection and discrimination of different polymer microparticles based on their characteristic Raman spectra was performed. Dextran, poly(vinyl alcohol), and melamine resin-based beads, with and without functionalization, were used for this purpose. Furthermore, taking advantage of the flow-through characteristics of the cell and the versatility of the employed flow system, full control over the media surrounding the trapped particles was achieved. This allowed us to perform chemical reactions on the trapped particles and to monitor spectral changes in real time. Here retention of cation-exchanger beads loaded with silver ions and subsequent reduction of the silver ions was demonstrated. In this way, surface-enhanced Raman (SER) active beads were prepared and retained in the focus of the Raman microscope by means of the ultrasonic field. Injection of analytes in the flow system thus allowed recording of their SER spectra. Using 9-aminoacridine, a linear dependence of the found SER signal in the range from 1 to 10 microM has been achieved. The repeatability in the recorded SER intensities was on the order of 4-5%. This included bead retention, surface-enhanced Raman layer synthesis, and analyte detection.     

Confocal Raman microscopy for monitoring chemical reactions on single optically trapped,solid-phase support particles

Optical trapping of small structures is a powerful tool for the manipulation and investigation of colloidal and particulate materials. The tight focus excitation requirements of optical trapping are well suited to confocal Raman microscopy. In this work, an inverted confocal Raman microscope is developed for studies of chemical reactions on single, optically trapped particles and applied to reactions used in solid-phase peptide synthesis. Optical trapping and levitation allow a particle to be moved away from the coverslip and into solution, avoiding fluorescence interference from the coverslip. More importantly, diffusion of reagents into the particle is not inhibited by a surface, so that reaction conditions mimic those of particles dispersed in solution. Optical trapping and levitation also maintain optical alignment, since the particle is centered laterally along the optical axis and within the focal plane of the objective, where both optical forces and light collection are maximized. Hour-long observations of chemical reactions on individual, trapped silica particles are reported. Using two-dimensional least-squares analysis methods, the Raman spectra collected during the course of a reaction can be resolved into component contributions. The resolved spectra of the time-varying species can be observed, as they bind to or cleave from the particle surface.     

Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli

Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid–protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.     

Sorting of micron-sized particles using holographic optical Raman tweezers in aqueous medium

We have constructed a holographic optical tweezers system combined with Raman spectroscopy to sort trapped particles. Our software automatically moves the trapped objects to the measurement positions to obtain individual Raman signals from multiple trapped particles. We performed the sorting by comparing their spectra with the previously measured training dataset using the correlation coefficients. We used yeast cells and polystyrene beads as test particles. This study aims to show that biological particles can be separated using single cell analysis with combined holographic optical tweezers and Raman spectroscopy system.     

Formation, stability, and mobility of one-dimensional lipid bilayers on polysilicon nanowires

Curved lipid membranes are ubiquitous in living systems and play an important role in many biological processes. To understand how curvature and lipid composition affect membrane formation and fluidity, we have assembled and studied mixed 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) supported lipid bilayers on amorphous silicon nanowires grown around carbon nanotube cores with controlled wire diameters ranging from 20 to 200 nm. We found that lipid vesicles fused onto nanowire substrates and formed continuous bilayers for all DOPC-DOPE mixtures tested (with the DOPE content of up to 30%). Our measurements demonstrate that nanowire-supported bilayers are mobile, exhibit fast recovery after photobleaching, and have a low concentration of defects. Lipid diffusion coefficients in these high-curvature tubular membranes are comparable to the values reported for flat supported bilayers and increase slightly with decreasing nanowire diameter. A free space diffusion model adequately describes the effect of bilayer curvature on the lipid mobility for nanowire substrates with diameters greater than 50 nm, but shows significant deviations from the experimental values for smaller diameter nanowires.     

Real-time detection of hyperosmotic stress response in optically trapped single yeast cells using Raman microspectroscopy

Living cells survive environmentally stressful conditions by initiating a stress response. We monitored changes in the Raman spectra of optically trapped Saccharomyces cerevisiae yeast cell under normal, heat-treated, and hyperosmotic stress conditions. It is shown that when glucose was used to exert hyperosmotic stress, two chemical substances-glycerol and ethanol-can be monitored in real time in a single cell.     

Optical trapping of unilamellar phospholipid vesicles: investigation of the effect of optical forces on the lipid membrane shape by confocal-Raman microscopy

Optical trapping of liposomes is a useful tool for manipulating these lipid vesicles for sampling, mechanical testing, spectroscopic observation, and chemical analysis. Through the use of confocal Raman microscopy, this study addresses the effects of optical forces on the structure of unilamellar, dipalmitoylphosphatidylcholine (DPPC) vesicles, both optically trapped in solution and adhered to a coverslip. The energy and forces involved in optical trapping of lipid vesicles were derived in terms of the dielectric contrast between the phospholipid membrane and the surrounding solution; reflection forces at the membrane/water interface were found to be negligible. At optical powers of 9 mW and greater, unilamellar liposomes trapped in bulk solution experience a gradient force sufficiently strong to bend the vesicle membrane, so that a second bilayer from the same vesicle is drawn into the optical trap, with an energy of approximately 6 x 10(-13) erg. For vesicles adhered to a coverslip, the confocal probe can be scanned through the attached vesicle. Optical forces are insufficient to detach the bilayer that is adhered to the glass; however, the upper DPPC bilayer can be manipulated by the optical trap and the shape of the vesicle distorted from a spherical geometry. The effect of calcium ion on the flexibility of membrane bilayers was also tested; with 5 mM calcium ion in solution, the lipid bilayer of a surface-attached liposome is sufficiently rigid so that it cannot be distorted at moderate laser powers.     

Comparison of the energetics of avidin, streptavidin, neutrAvidin, and anti-biotin antibody binding to biotinylated lipid bilayer examined by second-harmonic generation

A comparison of the binding properties of avidin, streptavidin, neutrAvidin, and antibiotin antibody to a biotinylated lipid bilayer was studied using second-harmonic generation. Protein binding assays were performed on a planar supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing 4 mol?% biotinylated-cap-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (biotin-cap-DOPE). The equilibrium binding affinities of these biotin-protein interactions were determined, revealing the relative energetic contributions for each protein to the biotinylated lipid ligand. The results show that the binding affinities of avidin, streptavidin, and neutrAvidin for biotin were all strengthened by protein-protein interactions but that the stronger protein-protein interactions observed for streptavidin and neutrAvidin make their binding more energetically favorable. It was also shown that neutrAvidin has the highest degree of nonspecific adsorption to a pure DOPC bilayer, compared to avidin and streptavidin. In addition, the biotin-binding affinity of the antibiotin antibody was found to be of the same order of magnitude as that of avidin, streptavidin, and neutrAvidin. These findings provide important new insights into these biotin-bound protein complexes commonly used in several bioanalytical applications.     

Lipid/Polyelectrolyte coatings to control carbon nanotubes intracellular distribution

Carbon Nanotubes have been functionalized with a layer of poly (sulfopropyl methacrylate) synthesized from silane initiators attached to the walls of the Carbon nanotubes. On top of the poly sulfo propyl methacrylate, lipid vesicles composed of 75% 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine and 25% 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] were assembled. The surface modification of the Carbon Nanotubes and lipid assembly were followed by TEM. Confocal Raman Microscopy was used to study the uptake and localization of the surface modified Carbon Nanotubes in the HepG2 cell line. The localization of the Carbon Nanotubes in the cells was affected by the surface coating. It was found that poly (sulfopropyl methacrylate) and lipid modified Carbon Nanotubes were present in the region of the lipid bodies in the cytoplasm.     

Parallel analysis of individual biological cells using multifocal laser tweezers Raman spectroscopy

We report on the development and characterization of a multifocal laser tweezers Raman spectroscopy (M-LTRS) technique for parallel Raman spectral acquisition of individual biological cells. Using a 785-nm diode laser and a time-sharing laser trapping scheme, multiple laser foci are generated to optically trap single polystyrene beads and suspension cells in a linear pattern. Raman signals from the trapped objects are simultaneously projected through the slit of a spectrometer and spatially resolved on a charge-coupled device (CCD) detector with minimal signal crosstalk between neighboring cells. By improving the rate of single-cell analysis, M-LTRS is expected to be a valuable method for studying single-cell dynamics of cell populations and for the development of high-throughput Raman based cytometers.     

Resonance-mode electrochemical impedance measurements of silicon dioxide supported lipid bilayer formation and ion channel mediated charge transport

A single-chip electrochemical method based on impedance measurements in resonance mode has been employed to study lipid monolayer and bilayer formation on hydrophobic alkanethiolate and SiO(2) substrates, respectively. The processes were monitored by temporally resolving changes in interfacial capacitance and resistance, revealing information about the rate of formation, coverage, and defect density (quality) of the layers at saturation. The resonance-based impedance measurements were shown to reveal significant differences in the layer formation process of bilayers made from (i) positively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), (ii) neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on SiO(2), and (iii) monolayers made from POEPC on hydrophobic alkanethiolate substrates. The observed responses were represented with an equivalent circuit, suggesting that the differences primarily originate from the presence of a conductive aqueous layer between the lipid bilayers and the SiO(2). In addition, by adding the ion channel gramicidin D to bilayers supported on SiO(2), channel-mediated charge transport could be measured with high sensitivity (resolution around 1 pA).