Joel M. Harris

Comparison of models describing the thermal lens effect.

A model for the thermal lens which approximates the refractive shape as a parabola is compared with a model which accounts for the aberrant nature of the lens; both models are tested against experimental results. The comparison suggests how the inaccuracies of the parabolic lens model may be corrected while retaining its mathematical simplicity and better predictive power for stronger thermal lenses.

Thermal lens calorimetry : Application to chromatographic detection

Abstract The laser-induced thermal lens effect has been applied to the calorimetric detection of absorbing samples having negligible fluorescence quantum yields. A simple but fast method for numerically fitting the thermal lens transient data, during the 0.25-sec period while the sample cools, allows the instrument to serve as a real-time absorbance monitor. Preliminary results, using 190 mW laser power, indicate detection limits of Amin ≈ 1.5·10-5 cm-1 for a 5-sec response time.

Biotin-avidin binding kinetics measured by single-molecule imaging.

The high affinity of avidin for biotin has made it useful for many bioanalytical applications involving the immobilization of proteins, vesicles, and other biomolecules to surfaces. To understand the formation and stability of the resulting biotin-avidin complex, it is useful to know the kinetics of the binding reaction, especially for situations where the complex is formed at a liquid-solid interface typically used in sensor or separation applications. In this work, a single-molecule fluorescence method is developed for measuring the kinetics and affinity constant for the binding of neutravidin, a deglycosylated variant of avidin, to surface-immobilized biotin. Biotin was immobilized using succinimidyl ester chemistry onto amine sites on glass surfaces. The surface density of biotin was controlled by the extreme dilution of 3-aminopropyltriethoxysilane into a monolayer of 2-cyanoethyltriethoxysilane. The resulting biotin binding sites are spaced apart by micrometer distances, and this avoids crowding effects and makes the resolution of single molecules possible. The binding and unbinding of individual tetramethylrhodamine-labeled neutravidin molecules is measured in situ by total-internal-reflection fluorescence (TIRF) microscopy imaging. Single-molecule detection and counting is readily achieved by this measurement, where quantitative control is established by determining the probabilities of false positive and negative events based on the intensity distributions of background and single-molecule spots and by comparing the bound molecule populations with the independently measured density of binding sites on the surface. The kinetics of binding and unbinding are evaluated by intermittent imaging and counting the number of bound neutravidin molecules versus time, following introduction of a neutravidin solution or its replacement by buffer over the low-density biotinylated surface. The neutravidin binding kinetics were found to be fast, essentially diffusion-controlled, while the stability of the complex and its dissociation rate appear to be influenced by the chemistry of biotin immobilization.

Room-temperature, excitation wavelength-dependent fluorescence at surfaces: a potential method for studying the micro-heterogeneity of surface environments

Abstract The fluorescence maxima at room temperature of 5-dimethylamino-1-naphthalenesulfonamide (dansylamide) groups covalently attached to the surface of silica particles in dry form and slurried in acetonitrile exhibit varying degrees of dependence upon excitation wavelength. This dependence is attributed to the micro-heterogeneity of the surface itself and varying degrees of accessibility of the fluorophore to solvent. The observed effect may prove useful in investigations of surface environment heterogeneity at the molecular level.

Total internal reflection fluorescence correlation spectroscopy for counting molecules at solid/liquid interfaces.

Fluorescence correlation spectroscopy, using tota internal reflection excitation (TIRFCS), is developed as a method to allow quantitative determination of molecular populations at solid/liquid interfaces. Population fluctuations of fluorescent molecules at the interface are observed as excess low-frequency noise on a fluorescence signal. Since the noise arises from molecular origins, its magnitude can be evaluated by Poisson statistics to determine the number of molecules in the interface volume. This quantitative information is available without sensitivity calibration or the preparation of standards and without fitting the transients to a kinetic model. Unlike single-molecule counting measurements, TIRFCS can produce these quantitative results even when the number of photoelectrons detected per molecule is small. Surface populations of rhodamine 6G dye molecules were measured at C-18-derivatized, flat silica surfaces in contact with aqueous solutions and compared with predicted values derived from chromatographic retention data. In addition, electrostatic and nonpolar contributions to the free energy of adsorption of the dye to C-18-modified silica surfaces were examined.

C18-modified metal-colloid substrates for surface-enhanced Raman detection of trace-level polycyclic aromatic hydrocarbons in aqueous solution

Metal colloids immobilized on a glass support substrate are modified with a self-assembled alkylsilane (C18) layer to promote adsorption of polycyclic aromatic hydrocarbons from aqueous solutions. Detection of these compounds from low concentration solutions is accomplished by using surface-enhanced Raman scattering (SERS). SERS spectra of pyrene adsorbed to C18-modified immobilized silver colloids are dominated by Raman bands that are not consistent with pyrene and indicate that pyrene undergoes a chemical reaction at the surface. The origins of this surface product are investigated, and it is determined that silver and oxygen are required to form the product, whose Raman spectrum is consistent with oxidation to a quinone. When a C18-modified gold-colloid substrate is used, Raman scattering consistent with unreacted pyrene is observed. The adsorption and detection of pyrene adsorbed from low (2 ppb) concentration aqueous solutions onto C18-modified gold-colloid substrates is reported; naphthalene and phenanthrene are detected at ∼5 ppb. Adsorption kinetics are rapid (<5 min), and the concentration-dependent SERS response is consistent with a Langmuir isotherm.

Electronic spectroscopic investigations of the stationary phase in reversed-phase liquid chromatography

Abstract Electronic spectroscopy of probe molecules provides a powerful means of characterizing the stationary phase in reversed-phase liquid chromatography. In particular, both fluorescence and UV—visible absorption spectroscopies have been used to characterize these complex interfacial environments. This article reviews the progress made with these approaches for studying the structure of the stationary phase, the solute environment that it produces, and the dynamics of sorbed molecules in reversed-phase liquid chromatography. Fluorescence studies using either covalently attached probes, or physiosorbed probes are reviewed, along with total internal reflection fluorescence studies of flat, model interfaces. Dynamic effects due to excimer formation and quenching are shown to provide information about hydrocarbon ligand proximity, microviscosity, and contact of sorbed molecules with the mobile phase. UV—visible diffuse reflectance spectroscopy has also been used to characterize the dipolarity, polarizability and hydrogen bonding interactions of the reversed-phase surface environment. These electronic spectroscopic approaches lend insight into the organization, orientation, and polarity of the alkyl chains. In this article, the results of these studies are reviewed, and their impact on models for reversed- phase retention are discussed.

Quantitative Detection of Single Molecules in Fluorescence Microscopy Images

Fluorescence imaging and counting of single molecules adsorbed or bound to surfaces are being employed in a number of quantitative analysis applications. Reliable molecular counts with knowledge of counting uncertainties, both false-positive and false-negative probabilities, are critical to these applications. By counting stationary single molecules on a surface, spatial criteria may be applied to the image analysis to improve confidence in detection, which is especially critical when detecting single fluorescent labels. In this work, we describe a simple approach to incorporating spatial criteria for counting single molecules by using an intensity threshold to locate regions with multiple, adjacent intense pixels, where the size of these regions is guided by the point-spread function of the microscope. By requiring multiple, spatially correlated bright pixels, false-positive events resulting from random samples of background noise are minimized. The reliability of detection is established by quantitative knowledge of the distributions of background and signals. By measuring and modeling both the background and single-molecule intensity distributions, false-positive and false-negative detection probabilities are estimated for arbitrary threshold parameters by using combinatorial statistics. From this theory, detection parameters can be optimized to minimize false-positive and false-negative probabilities, which can be calculated explicitly. For detection of single rhodamine 6G molecules at a threshold set at 2.5 times the standard deviation above background, the false-negative probability was only 1.5%, determined from distributions of single-molecule intensities on well-populated surfaces, and the false-positive probability from background noise was 2.8 spots per 50 x 50 microm image. The false-positive events compare favorably with theoretical probabilities calculated using combinatorial statistical analysis and simulated false-positive events counted in images of random noise.

Quantitative SERS measurements on dielectric-overcoated silver-island films by solution-deposition control of surface concentrations

A simple method to control the dosing of small adsorbate molecules onto solid surfaces from liquid solution is applied to quantitative surface-enhanced Raman scattering measurements on dielectric-overcoated silver-island films. The deposition method, based on substrate withdrawal from solution, is evaluated by measuring fluorescence (ex situ) and optical absorption (in situ) of dye molecules deposited onto glass surfaces. Control of adsorbate surface concentrations was accomplished by varying the withdrawal rate and the concentration of the dye in solution. The dosing method was used to study the dependence of the electromagnetic contribution to SERS enhancement on surface coverage of scatterer. The sensitivity enhancement was found to be constant for adsorbate coverages up to 60-80% of a monolayer. Beyond a full monolayer, SERS enhancement for additional molecules deposited onto the surface was found to drop significantly, by as much as 1 order of magnitude.

Single-molecule fluorescence imaging of peptide binding to supported lipid bilayers.

Single-molecule fluorescence imaging techniques have been adapted to the quantitative characterization of peptide-binding to lipid bilayers. Peptide-membrane interactions are important in therapeutics, diagnostics, and membrane permeation and for understanding of the structure and function of membrane-bound proteins. Total-internal reflection fluorescence (TIRF) imaging is capable of determining membrane-binding equilibrium constants through the reliable counting of individual peptide molecules in order to report their surface density in the membrane. The residence times of the individual molecules in the membrane can also be determined and the rates of unbinding determined from a histogram of residence times. A combination of the unbinding kinetics and the equilibrium constant allows the binding rate of a peptide to the membrane also to be reported. We apply this method to characterize the lipid membrane affinity of glucagon-like peptide-1 (GLP-1), a 30-residue membrane-active peptide that is involved in glycemic control. Using single-molecule TIRF imaging, we have measured the affiliation of GLP-1 with a supported, phospholipid bilayer and determined its binding equilibrium constant. Two rates of dissociation were observed, suggesting strongly and weakly bound states of the peptide. The rate of membrane association was much slower than diffusion-controlled, indicating a significant kinetic barrier to membrane binding. The data were interpreted using a heterogeneous, surface-reaction model analogous to electron-transfer kinetics at an electrode. To our knowledge, these results are the first example of using single-molecule counting to quantify peptide-lipid bilayer binding equilibria and kinetics.