Confocal Raman microscopy of optical-trapped particles in liquids.

The in situ analysis of small, dispersed particles in liquids is a challenging problem, the successful solution to which influences diverse applications of colloidal particles in materials science, synthetic chemistry, and molecular biology. Optical trapping of small particles with a tightly focused laser beam can be combined with confocal Raman microscopy to provide molecular structure information about individual, femtogram-sized particles in liquid samples. In this review, we consider the basic principles of combining optical trapping and confocal Raman spectroscopy, then survey the applications that have been developed through the combination of these techniques and their use in the analysis of particles dispersed in liquids.

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 spectroscopy-based metabolomics for differentiating exposures to triazole fungicides using rat urine.

Normal Raman spectroscopy was evaluated as a metabolomic tool for assessing the impacts of exposure to environmental contaminants, using rat urine collected during the course of a toxicological study. Specifically, one of three triazole fungicides, myclobutanil, propiconazole, or triadimefon, was administered daily via oral gavage to male Sprague-Dawley rats at doses of 300, 300, or 175 mg/kg, respectively. Urine was collected from all three treatment groups and also from vehicle control rats on day six, following five consecutive days of exposure. Spectra were acquired with a CCD-based dispersive Raman spectrometer, using 785-nm diode laser excitation. To optimize the signal-to-noise ratio, urine samples were filtered through a stirred ultrafiltration cell with a 500 nominal molecular weight limit filter to remove large, unwanted urine components that can degrade the spectrum via fluorescence. However, a subsequent investigation suggested that suitable spectra can be obtained in a high-throughput fashion, with little or no Raman-specific sample preparation. For the sake of comparison, a parallel 1H NMR-based metabolomic analysis was also conducted on the unfiltered samples. Results from multivariate data analysis demonstrated that the Raman method compares favorably with NMR in regard to the ability to differentiate responses from these three contaminants.

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.

Monitoring the speciation of aqueous free chlorine from pH 1 to 12 with Raman spectroscopy to determine the identity of the potent low-pH oxidant.

The speciation of aqueous free chlorine above pH 5 is a well-understood equilibrium of H2O + HOCl <==> OCl- + H3O+ with a pKa of 7.5. However, the identity of another very potent oxidant present at low pH (below 5) has been attributed by some researchers to Cl2(aq) and by others to H2OCl+. We have conducted a series of experiments designed to ascertain which of these two species is correct. First, using Raman spectroscopy, we found that an equilibrium of H2O + H2OCl+ <==> HOCl + H3O+ is unlikely because the "apparent pKa" increases monotonically from 1.25 to 2.11 as the analytical concentration is increased from 6.6 to 26.2 mM. Second, we found that significantly reducing the chloride ion concentration changed the Raman spectrum and also dramatically reduced the oxidation potency of the low-pH solution (as compared to solutions at the same pH that contained equimolar concentrations of Cl- and HOCl). The chloride ion concentration was not expected to impact an equilibrium of H2O + H2OCl+ <==> HOCl + H3O+, if it existed. These observations supported the following equilibrium as pH is decreased: Cl2(aq) + 2H2O <==> HOCl + Cl- + H3O+. The concentration-based equilibrium constant was estimated to be approximately 2.56 x 10(-4) M2 in solutions whose ionic strengths were approximately 0.01 M. The oxidative potency of the species in low pH solutions was investigated by monitoring the oxidation of secondary alcohols to ketones. These and other results reported here argue strongly that Cl2(aq) is the correct form of the potent low-pH oxidant in aqueous free-chlorine solutions.

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.

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.