Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (K d spanning six decades) and unmatched sensitivity (picomolar K d 's and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca 2+ , a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.

Optical contrast agents have been widely applied to enhance the sensitivity and specificity of optical imaging with near-infrared (NIR) light. However, because of the overwhelming scattering of light in biological tissues, the spatial resolution of traditional optical imaging degrades drastically as the imaging depth increases. Here, for the first time to our knowledge, we present noninvasive photoacoustic angiography of animal brains in vivo with NIR light and an optical contrast agent. When indocyanine green polyethylene glycol, a novel absorption dye with prolonged clearance, is injected into the circulatory system of a rat, it obviously enhances the absorption contrast between the blood vessels and the background tissues. Because NIR light can penetrate deep into the brain tissues through the skin and skull, we are able to successfully reconstruct the vascular distribution in the rat brain from the photoacoustic signals. On the basis of differential optical absorption with and without contrast enhancement, a photoacoustic angiograph of a rat brain is acquired that matches the anatomical photograph well and exhibits high spatial resolution and a much-reduced background. This new technology demonstrates the potential for dynamic and molecular biomedical imaging.

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Purpose:To evaluate noninvasive molecular imaging methods as correlative biomarkers of thera-

We describe a novel approach for measuring fluid bulk properties such as refractive index (RI) and temperature changes in tubes of capillary dimensions based on a simple optical configuration which uses an unfocused He-Ne laser, a cylindrical tube, and a photodetector. Side illumination of a fused silica capillary produces a fan radiation in a 360°plane normal to the central axis of the capillary that is spatially well defined in the lateral direction. It is shown that, upon viewing a high-contrast interference pattern contained in the beam profile on a flat imaging plane in a direct backscattering configuration, the physical characteristics of the capillary and the bulk properties of the fluid contained within the tube can be determined. Position changes in the maxima and minima in the intensity-modulated profile (interference fringes) are directly related to the refractive index of the fluid in the tube. Measurement of the fringe shift by a slitphotodetector assembly facilitates the determination of dn/n at the level of 1.8 × 10 -7 , while temperature changes can be measured using the fringe centroid position at a level of 5.8 × 10 -5 °C (DL ) 3σ). Each determination can be made within a probe volume of 2.6 nL. Also, a wide range of tube diameters can be employed (75-775 µm i.d.) with no modification to the optical train or measurable degradation in system performance. We report results for an optical ray trace model for predicting fringe pattern energy distributions, optimization of the optical configuration, and general system performance predictions. Comparison of our four-beam interference model with experimental results illustrates the utility of the model.

Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show backscattering interferometry (BSI) to be a viable technique for quantifying ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic- and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity, and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest.

An on-chip detector based on backscatter interferometry has been developed to perform subnanoliter-volume refractive index measurements. The detection system consists of a simple, folded optical train based on the interaction of a laser beam and an etched channel, consisting of two radii joined by a flat portion, thus defining a curved surface in the shape of a hemisphere in a silica (glass) plate. The backscattered light from the channel takes on the form of a high-contrast interference pattern that contains information related to the bulk properties of the fluid contained within the probe volume. Positional changes of the interference pattern (fringes) allow for the determination of deltan at the 10(-6) level, corresponding to 743 microM or 139 x 10(-15) mol or 12.8 x 10(-12) g of sucrose, in a probe volume of only 188 x 10(-12) L. A theoretical model of the on-chip backscatter interferometric detector has also been developed, evaluated, and found to be in agreement with experimental data. It is shown that the model can be used to predict general system performance for changes in the optical train such as the chip's wall thickness and channel diameter.

Quantification of protein-protein and ligand-substrate interactions is central to understanding basic cellular function and for evaluating therapeutics. To mimic biological conditions, such studies are best executed without modifying the proteins or ligands (i.e., label-free). While tools for label-free assays exist, they have limitations making them difficult to fully integrate into microfluidic devices. Furthermore, it has been problematic to reduce detection volumes for on-channel universal analyte quantification without compromising sensitivity, as needed in label-free methods. Here we show how backscattering interferometry in rectangular channels (BIRC) facilitates label-free studies within picoliter volumes. The simple and unique optical train was based on rectangular microfluidic channels molded in poly(dimethylsiloxane) and low-power coherent radiation. Quantification of irreversible streptavidin-biotin binding and reversible protein A-human IgG Fc molecular interactions in a 225 pL detection volume was carried out label-free and noninvasively. Detection limits of 47 x 10(-15) mol of biotin reacted with surface-immobilized streptavidin were achieved. In the case of reversible interactions of protein A and the Fc fragment of human IgG, detection limits were determined to be 2 x 10(-15) mol of IgG Fc. These experiments demonstrate for the first time that (1) high-sensitivity universal solute quantification is possible using interferometry performed within micrometer-sized channels formed in inexpensive PDMS chips, (2) label-free reversible molecular interaction can be studied with femtomoles of solute, and (3) BIRC has the potential to quantify binding affinities in a high-throughput format.