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.     

Measurement of membrane rigidity on trapped unilamellar phospholipid vesicles by using differential confocal microscopy

We use optical tweezers to trap a unilamellar phospholipid vesicle and measure the out-of-plane thermal fluctuations by using differential confocal microscopy. Bending moduli of the lipid membranes are calculated directly from the mean-square values of the fluctuation amplitudes. Owing to the refractive index contrast between the inner and outer solutions of the vesicle, optical tweezers trap the vesicle laterally and improve the reliability of the measured fluctuation amplitudes along the optical axis. Bending moduli of membranes in gel or fluid phases obtained by the combination of differential confocal microscopy and optical tweezers are close to those reported previously. We also obtain the bending modulus of sphingomyelin membranes in the gel phase, which was not reported previously.     

Imaging theory of nonlinear Raman confocal microscopy

A novel nonlinear Raman confocal microscopy utilizing Raman induced Kerr effect spectroscopy (RIKES) is presented in this paper. The imaging theory of RIKES confocal microscopy with Gaussian beam is derived. The imaging properties of RIKES confocal microscopy and the impact of different beam waist widths of Gaussian beam on the lateral and axial resolution have been analyzed in detail. It is proved that RIKES confocal microscopy has high sensitivity and high resolution, besides capability to characterize inherent structural features, such as vibration mode, vibration orientation, and optically induced molecular reorientation etc. Therefore, nonlinear Raman confocal microscopy that is based on RIKES has potential to provide a novel characteristic imaging method comparable to the existing imaging techniques based on other nonlinear optical processes, such as two-photon fluorescence, second harmonic generation (SHG) and coherent anti-Stoke Raman scattering (CARS).     

Quantitative fluorescence microscopy to determine molecular occupancy of phospholipid vesicles

Encapsulation of molecules in phospholipid vesicles provides unique opportunities to study chemical reactions in small volumes as well as the behavior of individual proteins, enzymes, and ribozymes in a confined region without requiring a tether to immobilize the molecule to a surface. These experiments generally depend on generating a predictable loading of vesicles with small numbers of target molecules and thus raise a significant measurement challenge, namely, to quantify molecular occupancy of vesicles at the single-molecule level. In this work, we describe an imaging experiment to measure the time-dependent fluorescence from individual dye molecules encapsulated in ~130 nm vesicles that are adhered to a glass surface. For determining a fit of the molecular occupancy data to a Poisson model, it is critical to count empty vesicles in the population since these dominate the sample when the mean occupancy is small, λ ≤ ~1. Counting empty vesicles was accomplished by subsequently labeling all the vesicles with a lipophilic dye and reimaging the sample. By counting both the empty vesicles and those containing fluors, and quantifying the number of fluors present, we demonstrate a self-consistent Poisson distribution of molecular occupancy for well-solvated molecules, as well as anomalies due to aggregation of dye, which can arise even at very low solution concentrations. By observation of many vesicles in parallel in an image, this approach provides quantitative information about the distribution of molecular occupancy in a population of vesicles.     

Quantitative phase imaging in confocal microscopy by optical differentiation

The technique of optical differentiation is applied to confocal microscopy for the purpose of quantitative phase imaging. One-dimensional absorptive filters are placed in the pupil of the microscope objective to produce images related to the local phase slope in the object. With suitable signal processing and integration, a quantitative phase profile is obtained. This method is demonstrated in a reflection-based surface-profiling instrument.     

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.     

Apertureless tip-enhanced Raman microscopy with confocal epi-illumination/collection optics

It is demonstrated that confocal epi-illumination/collection optics can be effectively used to generate surface-enhanced Raman scattering at the near-field region of a gold-coated tip for an atomic force microscope operated in semi-contact tapping mode. When the tip, with a 50-nm apex radius, was illuminated by a highly focused laser beam at 532 nm and approached the isolated diamond particle, with a size of approximately 1 microm, the Raman signal was enhanced by approximately 10(3). This result is in good agreement with numerical simulations performed by the finite difference time-domain method. Since our apertureless microscope is based on readily available conventional components, there is wide room for improvements and modifications by common users in various applications of micro-Raman analysis.     

Ligand distributions in agarose particles as determined by confocal Raman spectroscopy and confocal scanning laser microscopy

Confocal Raman spectroscopy and confocal scanning laser microscopy have been used to analyze ligand distributions within individual chromatographic adsorbent particles. Three different types of particles have been investigated. The first type was synthesized to have a uniform distribution of allyl groups, whereas the two others were designed to have a surface layer of sulphopropyl groups and cores containing allyl groups and dextran, respectively. With confocal Raman spectroscopy it was possible to follow the distribution of both the surface layer and the interior. The distribution of sulphopropyl groups was evaluated with both confocal scanning laser microscopy and confocal Raman spectroscopy, whereas the distributions of allyl groups and dextran were evaluated only with the latter method. The results from the confocal measurements showed the expected result with a uniform distribution of allyl groups in the first type of particle and surface layers of sulphopropyl groups and cores with dextran or allyl groups for the two others.     

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.     

Optimizing detection sensitivity on surface-enhanced Raman scattering of transition-metal electrodes with confocal Raman microscopy

Some points on how to improve the detection sensitivity of confocal Raman microscopy for the study of surface-enhanced Raman scattering (SERS) of transition-metal electrodes are discussed, including the careful design of the spectroelectrochemical cell, proper selection of the thickness of the solution layer, the binning of charge-coupled device (CCD) pixels, and appropriate setting of the notch filter. Various roughening methods for the Pt, Rh, Fe, Co, and Ni electrode surfaces have been introduced in order to obtain SERS-active surfaces. It has been shown that the appropriate roughening procedure and the optimizing performance of the confocal Raman microscope are the two most important factors to directly generate and observe SERS on net transition-metal electrodes.     

Spatially resolved characterization of cellulose nanocrystal-polypropylene composite by confocal Raman microscopy

Raman spectroscopy was used to analyze cellulose nanocrystal (CNC) -polypropylene (PP) composites and to investigate the spatial distribution of CNCs in extruded composite filaments. Three composites were made from two forms of nanocellulose (CNCs from wood pulp and the nano-scale fraction of microcrystalline cellulose) and two of the three composites investigated used maleated PP as a coupling agent. Raman maps, based on cellulose and PP bands at 1098 and 1460 cm(-1), respectively, obtained at 1 μm spatial resolution showed that the CNCs were aggregated to various degrees in the PP matrix. Of the three composites analyzed, two showed clear existence of phase-separated regions: Raman images with strong PP and absent/weak cellulose or vice versa. For the third composite, the situation was slightly improved but a clear transition interface between the PP-abundant and CNC-abundant regions was observed, indicating that the CNC remained poorly dispersed. The spectroscopic approach to investigating spatial distribution of the composite components was helpful in evaluating CNC dispersion in the composite at the microscopic level, which helped explain the relatively modest reinforcement of PP by the CNCs.     

Spectroscopic microscopy analysis of the interior pH of individual phospholipid vesicles

The use of phospholipid vesicles as reaction containers, as vehicles for pharmaceutical drug delivery, and as model systems for cells has prompted the development of new methods for analyzing the structure of vesicles and their contents. The pH of the interior of vesicles is of particular interest when analytes are encapsulated and concentrated with the use of a pH gradient. While the interior pH is generally measured for large populations of vesicles, we report the measurement of the interior pH of individual vesicles as their buffer contents are titrated by transfer of N-methylbutylamine (NMBA) into the vesicle by a pH gradient. The initially acidic buffer within the vesicles is titrated along with a small concentration of an encapsulated pH sensitive dye, 5,6-carboxy SNARF-1-dextran. Images of the indicator fluorescence from each vesicle and its dispersed fluorescence spectrum are recorded using epi-illumination spectral fluorescence microscopy. From a fit of the spectra to the respective acid and base forms of the fluorescent indicator, the interior pH of individual vesicles as a function of the concentration of the NMBA titrant in the external solution could be determined.     

Characterization of a hazardous eyeliner (kohl) by confocal Raman microscopy

A new method of analyzing kohl, a cosmetic eyeliner, using confocal Raman microscopy is reported. This technique offers an important alternative to conventional spectroscopic techniques that provide elemental/atomic composition. Raman spectra of three kohl samples have been measured between 150 and 3000 cm(-1) at room temperature. The main component of two kohl samples was found to be lead(II) sulfide (PbS). Kohl is used as a traditional cosmetic and remedy in the Middle East, Far East, and Northern Africa. Since kohl products contain very high concentrations of lead, they constitute a risk for public health, particularly for children.     

Measuring diffusion of molecules into individual polymer particles by confocal Raman microscopy

Raman microscopy is a powerful method for providing spatially resolved, chemically selective information about the composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles in contact with liquid solutions. In this work, the transport of an organic solvent component into small polystyrene particles is investigated. An inverted confocal Raman microscope is used to acquire spectra from individual 75-microm polystyrene particles in contact with acetonitrile/water mixtures. Monitoring the Raman scattering from the C[triple bond]N stretching mode of acetonitrile provides a measure of solvent uptake into the polymer material. The small collection volume defined by the confocal optics provides the micrometer spatial resolution needed to track solvent concentration at different locations within the particle with 30-s time resolution. The volume fraction of acetonitrile in water in the surrounding solution was varied in order to determine the concentration dependence of the diffusion kinetics. Modeling the transport of molecules into a particle was addressed by using finite element methods for the evaluation of the coupled space- and time-dependent differential equations that govern the molecular transport. The results indicate that the diffusion coefficient changes with the local solvent concentration in the polymer. At longer times, with the highest acetonitrile concentrations, an evolution of the solvent transport mechanism was observed, from a diffusive rate that depends on local concentration to a linear increase in concentration with time accompanied by measurable swelling of the particle volume.     

Hybrid confocal Raman fluorescence microscopy on single cells using semiconductor quantum dots

We have overcome the traditional incompatibility of Raman microscopy with fluorescence microscopy by exploiting the optical properties of semiconductor fluorescent quantum dots (QDs). Here we present a hybrid Raman fluorescence spectral imaging approach for single-cell microscopy applications. We show that resonant Raman imaging of flavocytochrome b558 at 413.1 nm excitation in QD-labeled neutrophilic granulocytes or nonresonant Raman imaging of proteins and lipids at 647.1 nm excitation in QD-labeled macrophages can be integrated with linear one-photon excitation and nonlinear continuous-wave two-photon excitation fluorescence microscopy of QDs, respectively. The enhanced information content of these two hybrid Raman fluorescence methods provides new multiplexing possibilities for single-cell optical microscopy and intracellular chemical analysis.     

Depth characterization of photopolymerized films by confocal Raman microscopy using an immersion objective

The depth characterization of photopolymer films by confocal Raman microscopy is often troublesome due to refraction effects. To minimize these effects, we used an oil immersion objective and a method was developed to avoid penetration of the oil without damaging the sample surface. Since the surface may be sticky if oxygen in the air inhibits the photopolymerization, a protective layer could not be put onto the film. Therefore, the method consists in using a thin polypropylene foil as substrate for the coating and placing the sample upside down under the objective. In this manner, the immersion oil could be deposited on top of the polypropylene. The advantage of this setup is that the oil, polypropylene substrate, and photopolymer film have close refractive indices. Basic calculations showed that the depth resolution is hardly affected in that configuration and double-bond conversion profiles could be plotted as a function of reliable nominal depth. The validity of the methodology was confirmed by experiments carried out with a dry metallurgical objective on the sample surface, face up, where refraction effects are still minor. In addition, infrared spectroscopy, which was used to follow the photopolymerization, corroborated the Raman conversion of the films over their thickness. The confocal Raman microscopy method can be applied to various photopolymerized systems to characterize their behavior towards oxygen inhibition and other heterogeneities in conversion arising from inner filter effects or interactions between additives for instance.     

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.     

Multivariate analysis applied to the study of spatial distributions found in drug-eluting stent coatings by confocal Raman microscopy

Multivariate data analysis was applied to confocal Raman measurements on stents coated with the polymers and drug used in the CYPHER Sirolimus-eluting Coronary Stents. Partial least-squares (PLS) regression was used to establish three independent calibration curves for the coating constituents: sirolimus, poly(n-butyl methacrylate) [PBMA], and poly(ethylene-co-vinyl acetate) [PEVA]. The PLS calibrations were based on average spectra generated from each spatial location profiled. The PLS models were tested on six unknown stent samples to assess accuracy and precision. The wt % difference between PLS predictions and laboratory assay values for sirolimus was less than 1 wt % for the composite of the six unknowns, while the polymer models were estimated to be less than 0.5 wt % difference for the combined samples. The linearity and specificity of the three PLS models were also demonstrated with the three PLS models. In contrast to earlier univariate models, the PLS models achieved mass balance with better accuracy. This analysis was extended to evaluate the spatial distribution of the three constituents. Quantitative bitmap images of drug-eluting stent coatings are presented for the first time to assess the local distribution of components.     

Comparison of discrete and continuous motion in scanning probe microscopy monitored via confocal Raman microspectroscopy

Confocal Raman imaging is a relatively new analytical technique that combines the strengths of Raman microspectroscopy and confocal optics. The images collected by the microscope are obtained by monitoring specific bands in the Raman spectra that are collected at many points in a sample, with the number of spectra usually numbering in the hundreds or thousands. Some commercially available systems acquire data while the sample is continuously moving with respect to the microscope objective. The distance that the stage moves during a single acquisition is a parameter that can be set prior to data acquisition. Data in this report was acquired with both a static and continuously moving sample for comparison, utilizing the 520 cm(-1) Si phonon of a silicon wafer to monitor an edge. Scattering collected from each discrete step, i.e., no motion during spectral acquisition, showed excellent precision of location, but a loss in resolution was observed as the pixel size was increased beyond the maximum theoretical resolution of the instrument. A continuously moving stage contributed to erroneous position data as the pixel size was increased beyond the maximum theoretical resolution of the instrument.     

Raman spectroscopic studies of the phase transitions in hexane at high pressure

Raman spectroscopic study of n-hexane was carried out in a cubic zirconia anvil cell up to approximately 2.0 GPa. Under high pressure, the C-H stretching region of the spectrum at 2850-3000 cm(-1) shows measurable changes in frequency, bandwidth, and intensity. These Raman bands shift towards higher frequencies with increasing pressure. At about 1.4 GPa, phase transition from liquid to solid was induced by compression, as was simultaneously observed with the built-in microscope.