The conventional atomic force microscope (AFM) is equipped with a single optical detection system. Probe-sample separation is determined in an independent deflection with respect to AFM z-translation experiment. This method of determining probe-surface separation is relative, susceptible to drift and does not provide real time separation information. The evanescent wave atomic force microscope (EW-AFM) utilizes a second, independent detection system to determine absolute probe-surface separation in real time. The EW-AFM can simultaneously acquire real-time force and probe-sample separation information using the optical lever and evanescent scattering detection systems, respectively. The EW-AFM may be configured with feedback on the optical-lever system for constant force applications or with feedback on evanescent wave scattering intensity for constant height applications. Scattering of the evanescent wave exponential decay profile is used to determine probe-surface separation. Sub-micron sized dielectric and metallic probes show exponential scattering profiles, micron sized polystyrene and borosilicate microspheres show non-exponential profiles when they are affixed beneath the cantilever tip. By affixing the microspheres to the end of the AFM cantilever exponential and non-exponential profiles were observed. The EW-AFM can be used to conduct force-distance and imaging experiments. The EW-AFM was used to measure the thickness of surfactant bilayers formed at the silica-solution interface using silicon nitride AFM tips. The presence of a refractive index difference between the surfactant bilayer and the solution does not influence the accuracy of the surfactant bilayer thickness measurement. The EW-AFM was used to scan a 2 x 2 micron area in constant height mode. The probe was brought to within 6 nanometers of a planar dielectric surface using the evanescent wave intensity as a height reference with accuracy of ± 1 nm. This capability may be utilized to observe charge heterogeneity at the solid-liquid interface with nanometer lateral resolution or to map chemical functional group heterogeneity based on perturbations to the electrical double layer. The EW-AFM evanescent scattering system has an absolute separation resolution of 0.3 nm compared to 1.0 nm relative separation resolution for the optical lever system. In constant scattering (constant height) mode the real time separation precision is about 2 nm. / Ph. D.

We operated focus ion beam instrument (FIB) to etch array of cylindrical holes, with the diameter smaller than the wavelength of visible, on the sliver film. The sample was probed with the tungsten light source and the transmission spectrum of visible-NIR range was recorded with traix550 spectrometer with a cooling CCD. The optical extraordinary properties, such as the light can be transmitted through the holes array with the diameter of the hole smaller than the wavelength of the incident light and the enhancement of the efficiency, of the transmission of the subwavelength holes array were discussed. In this study, the factors affected the transmission spectrum of the subwavelength hole array, e.g.: the arrangement of the holes array, the material of the thin film, the diameter of the hole¡Kand so on, were researched. Besides, we modulated the effective dielectric property of the cavity of the subwavelength holes with the thermal evaporation method. It was pointed out that effective dielectric property of the cavity also influenced the optical properties of extraordinary transmission spectrum. We believe that our research was helpful to understand the physical properties of the subwavelength holes array.

The motion of dye molecules in the solution is highly influenced by the Brownian motion caused by the stochastic collisions with the solvents, and it results the fluorescence intensity fluctuation. The thesis study the fluorescence intensity fluctuation of dilute dye molecule (Rhodamine B) in methanol solution ( - ), under confocal and total internal reflection (TIR) microscopy configurations. Five parameters are used to probe the fluorescence characteristics: (1) the difference between confocal and the TIR configurations. The configuration influences the laser focusing area and consequently the intensity distribution. The effective focusing area in confocal configuration is an ellipsoid shape, while that of TIR configuration is a disk shape around the interface with depth 100-200 nm. It results the TIR configuration less background and higher concentration capability. (2) concentration. We control the concentration from much less than one molecule to more than one molecule in the effective focusing area, and we observe the change of burst intensity distribution. (3) the focus position. By changing the focusing position, we study the effective focusing region changes. (4) excited intensity, and (5) fluorescence correlation spectroscopy (FCS). Our results indicate that TIR configuration exhibits lower background, and is suitable to higher concentration solution. In addition, when the dye concentration in the focusing area is much less than 1, the FCS amplitude is no longer follow 1/N, but rather be proportional to N, where N is the concentration.

An optical fiber humidity sensor has been devised using a porous sol-gel silica (PSGS) coating as a transducer. Evanescent wave scattering (EWS) in the PSGS coating. PSGS particles are highly hydrophilic and have a strong tendency to absorb water molecules from the surrounding environment. The absorbed water molecules form a thin layer on the inner surface of the pores inside the porous silica and enhance EWS, from which an indicatory signal can be obtained. The humidity sensor presented in this thesis has a fast response, is reversible, low cost, and has a broad dynamic relative humidity range from 3.6?0-6% to 100% or humidity range from 1.2ppm to 30000ppm. Because of its multiple advantages, including immunity to electromagnetic interference, resistance to corrosive environments, and high sensitivity, this humidity sensor has various applications. In soil moisture sensing, this humidity sensor can avoid the interference caused by compounds in soil water. For electrical transformer moisture sensing, this humidity sensor can avoid the effect of electromagnetic fields.

This thesis will characterize the use of HFAPNB and PHOST as a sensing layer material for a fully integrated multi-mode interferometric evanescent waveguide. The compatibility of HFAPNB and PHOST with standard CMOS and MEMS processing allowed us to explore their characteristics for sensing different analytes which included water, methanol, isopropyl alcohol, and benzene. The relative sensitivity of the polymer HFAPNB and PHOST to different chemicals has been quantified and related to a refractive index shift and thickness change of the polymers. Diffusion coefficients have also been quantified for both polymers and interaction models for each polymer and analyte has been determined to predict the concentration of a mixture of analytes.

This dissertation describes new methods for measuring surface forces using evanescent waves, and applications to non-DLVO forces. An evanescent wave, generated at a solid-liquid interface, is scattered by AFM tips or particles attached to AFM cantilevers. The scattering of this wave is used to determine absolute separation between surfaces and/or the refractive index as a function of separation in AFM measurements. This technique is known as evanescent wave atomic force microscopy (EW-AFM). The scattering of an evanescent wave by Si3N4 AFM tips is large and decays exponentially with separation from a refractive index boundary. Thus, scattering is a useful method for measuring the separation between a Si3N4 tip and sample. This method has been used to measure the absolute separation between a tip and sample in the presence of an irreversibly adsorbed polymer film. Measurement of the film thickness and time response of the polymer to applied loads has also been studied. These measurements are not possible using current AFM techniques. In addition to measurements in polymer systems, the simple scattering profile from Si3N4 tips was used to re-examine short range hydration forces between hydrophilic surfaces. Results presented in this thesis suggest this force does not depend on the hydrated radius of the ion between glass and silicon nitride. The scattering generated by a Si3N4 tip has also been used to measure the refractive index of bulk fluids and thin films between hydrophobic surfaces. Based on these results, I have shown that a long-range attraction between hydrophobic surfaces is accompanied by an increase in the refractive index between the tip and surface. From this I have concluded that the attractive force, measured in this study, is the result of an increase in the concentration of organic material between surfaces. Finally, I have shown that the scattering profile depends on the material and size of the scattering object. Scattering from silicon nitride tips is exponential with separation. In contrast, the scattering profile from silicon tips, which are similar in size and geometry, is not a simple exponential. The scattering profile of larger spherical particles attached to cantilevers is also not exponential. It is approximately the sum of two exponentials. The functional form of the scattering profile with separation is consistent with the transmission of evanescent light through flat planar films. This result would suggest that a re-examination of the separation-dependence of scattering in TIRM measurements is necessary. / Ph. D.

Biosensors are detection devices that couple biological recognition elements to physiochemical transducers to generate quantifiable signals. Immunosensors are biosensors that use antibodies as the recognition element. The highly specific nature of antibody-antigen binding is exploited to create immunosensors that are sensitive to analytes in complex mixtures and demonstrate a rapid response. Fiber optical immunosensors based on long-period gratings have limited sensitivity at the refractive index of ordinary aqueous solutions (~1.33). A composite film was designed to raise the local refractive index of the sensor, thus increasing sensitivity. Titanium dioxide deposition raised the refractive index of the sensor to ~1.42. Bovine serum albumin was immobilized onto a dextran hydrogel and attached to the LPG element via reductive amination. The thickness of the hydrogel was estimated to be 500 nm using Environmental Scanning Electron Microscopy. The affinity film was probed by an evanescent wave to detect changes in refractive index due to the binding of anti-BSA IgG. Under these conditions, the sensor yielded a signal ratio of approximately 10-4 refractive index units per nm signal. Reproducible binding was shown over multiple exposures, with no cross reactivity for non-specific antibodies and other proteins. Anti-BSA IgG (20 µg/mL) in whole serum was recycled through the fiber holder with an accompanying peak wavelength shift that averaged 2 nm on an Optical Spectrum Analyzer with a noise level of 0.1 nm. The BSA affinity film was regenerated 50 times and showed a baseline shift of -1.3 nm. / Master of Science

Robust ion-exchanged glass waveguides exhibit low optical losses in a broad spectral range and they allow integration of several devices on the same chip due to their planar structure. Consequently, they can be a low cost alternative to semiconductors for fabricating various integrated optical devices. Two high performance photonic devices were designed and realized, demonstrating the potential of glass waveguides. The well-controlled silver-film ion-exchange process allowed the fabrication of: i) a highly sensitive biosensor based on optical absorption and, ii) a low loss hybrid electro-optic (EO) polymer modulator with a narrow coplanar electrode gap. The single-mode, channel integrated optical ion-exchange waveguide on borosilicate glass (Corning 0211) is described for broad spectral band (400-650 nm) detection and analysis of heme-containing protein films at a glass/water interface. The evanescent wave interaction is improved significantly by fabricating ion-exchange waveguides with a step-like index profile. Silver nano-particle formation is reduced in order to achieve low loss in the Soret-band (~400 nm). Unlike other surface-specific techniques (e.g. SPR, interferometry) that probe local refractive-index changes and therefore are susceptible to temperature fluctuations, the integrated optical waveguide absorption technique probes molecular-specific transition bands and is expected to be less vulnerable to environmental perturbations. The hybrid integration of phosphate glass (IOG-1) and EO polymer is realized for the first time. The critical alignment steps which are typically required for hybrid optoelectronic devices are eliminated with a simple alignment-free fabrication technique. The low loss adiabatic transition from glass to EO polymer waveguide is enabled by gray scale patterning of the novel EO polymer, AJLY. Total insertion loss of 5 dB and electrode gap of 8 μm is obtained for an optimized device design. EO polymer poling at 135 ºC and 75 V/μm is enabled by the sol-gel buffer layer.

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) is an important signaling molecule as substrate for the phospholipase C (PLC)-catalyzed formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, and by directly regulating e.g. ion-channels, the cytoskeleton and vesicle trafficking in various types of cells. The present studies provide insights into the regulation of PLC activity and the plasma membrane concentration of PIP2 in individual insulin-secreting cells. Real-time monitoring of plasma membrane PIP2 was performed with evanescent wave microscopy and the PIP2/IP3-binding pleckstrin-homology-domain from PLC-δ1 fused to GFP. It was demonstrated that membrane depolarization and voltage-dependent Ca2+ influx are sufficient to activate PLC. Rise of the glucose concentration triggered Ca2+-dependent activation of PLC. Simultaneous measurements of the cytoplasmic Ca2+ concentration ([Ca2+]i) demonstrated that oscillations of [Ca2+]i resulting from periodic influx induced cyclic activation of PLC. Activation of muscarinic receptors caused a biphasic PLC response with an initial peak enhanced by positive feedback by Ca2+ mobilized from intracellular stores, followed by sustained activity depending on store-operated Ca2+-entry. Activation of PLC by Ca2+ mobilized from intracellular stores was part of the Ca2+-induced Ca2+ release mechanism by which glucagon stimulates primary mouse pancreatic β-cells. Experiments in permeabilized cells demonstrated rapid turnover of PIP2 with t1/2 ~ 16s. ATP stimulated concentration-dependent synthesis of plasma membrane PIP2, counteracted by the ADP analogue ADPβS. RT-PCR analysis identified transcripts of 10 different phosphoinositide-kinases. The ATP-stimulated PIP2 formation was mediated by type II and III PI4-kinases as well as by PIP5-kinase Iβ. It is concluded that the PIP2 concentration in the plasma membrane is regulated by the ATP/ADP ratio and that its hydrolysis by PLC is tightly controlled by [Ca2+]i in insulin-secreting cells.

cAMP and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting pancreatic β-cells. Glucose-stimulated insulin secretion from β-cells is pulsatile and driven by oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the kinetics of cAMP signaling and the mechanisms of cAMP action. Evanescent wave microscopy and fluorescent translocation biosensors were used to monitor plasma membrane-related signaling events in single MIN6-cells and primary mouse β-cells. Glucose stimulation of insulin secretion resulted in pronounced oscillations of the membrane phospholipid PIP3 caused by autocrine activation of insulin receptors. Glucose also triggered oscillations of the sub-plasma membrane cAMP concentration ([cAMP]pm). These oscillations were preceded and enhanced by elevations of [Ca2+]i, but conditions raising cytoplasmic ATP triggered [cAMP]pm elevations without accompanying changes in [Ca2+]i. The [cAMP]pm oscillations were also synchronized with PIP3 oscillations and both signals were suppressed after inhibition of adenylyl cyclases. Protein kinase A (PKA) was important for promoting concomitant initial elevations of [cAMP]pm and [Ca2+]i, and PKA inhibitors diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The glucose-induced PIP3 oscillations were markedly suppressed in cells treated with siRNA against the cAMP-dependent guanine nucleotide exchange factor Epac2. Pharmacological activation of Epac restored PIP3 responses after adenylyl cyclase or PKA inhibition. Glucose and other cAMP-elevating stimuli induced redistribution of fluorescence-tagged Epac2 from the cytoplasm to the plasma membrane. This translocation was modulated by [Ca2+]i and depended on intact cyclic nucleotide-binding and Ras-association domains. In conclusion, glucose generates cAMP oscillations in β-cells via a concerted action of Ca2+ and metabolically generated ATP. The oscillations are important for the magnitude and kinetics of insulin secretion. While both protein kinase A and Epac is required for initiation of insulin secretion the cAMP-dependence of established pulsatility is mediated by Epac2.