This work involves the development of a new technique that integrates Diffusing Colloidal Probe Microscopy (DCPM) and luminescence to simultaneously measure multiple particle-wall interactions. DCPM can be used to map potential energy profiles of multiple particle-surface interactions simultaneously and accurately. Colloidal semiconductor quantum dots were used for spectral multiplexing to enable monitoring of multiple analytes at the same time. DCPM combines Total Internal Reflection Microscopy (TIRM) and Video Microscopy to simultaneously measure multiple particle-surface interactions with nanometer resolution in particle-surface separation. By acquiring the scattered intensity emitted by the particles, the separation distance can be calculated and subsequently the forces of interactions between the particle and the surface. This work demonstrates the use of luminescence instead of scattering as the mode of detection in DCPM. The luminescence is provided by quantum dots which are incorporated into polystyrene microspheres. The unique optical properties of quantum dots enable the creation of an optically multiplexed system where microspheres are tagged by quantum dots of different emission wavelengths. Scattering in DCPM may result in erroneous calculation of the potential energy profiles because of particle polydispersity. Since scattering is dependent on particle size, luminescence is introduced into the system and some interesting results are obtained. These results illustrate that the effect of particle polydispersity is significantly reduced when luminescence is used as the mode of detection. This combined with the DCPM system’s sensitivity would enable the monitoring of multiple functionalized particlesurface interactions simultaneously and accurately.

Single particle TIRM experiments measure particle-surface separation distance by tracking scattered intensities. The scattered light is generated by an evanescent wave interacting with a levitating microsphere. The exponential decay of the evanescent wave, normal to the surface, results in scattered intensities that vary with separation distance. Measurement of the separation distance allows us to calculate the total potential energy profile acting on the particles. These experiments have been shown to exhibit nanometer spatial resolution and the ability to detect potentials on the order of kT with no external treatment of the particle. We find that the separation distance is a function of the decay of the evanescent wave and the size of the sphere. Different sizes of spheres, located the same distance from the surface, exhibit varying scattered intensity distributions. Single particles have been studied extensively but multiple particle experiments are needed for studies of more complex systems and surfaces. Increasing the number of colloidal particles in a TIRM experiment greatly increases the complexity of the system. Calculation of separation distances and potentials over a large group of microspheres requires that the spheres display a uniform stuck-particle intensity distribution. But, for large numbers of particles, this is not the case. In some instances, stuck-particle intensities can vary more than an order of magnitude. This research involves creating a mathematical model to study scattered intensity distributions for a large size range of polystyrene microspheres. The model is based on basic Mie theory. We compare the theoretically simulated results to the experimentally obtained results and find that scattered intensity variations in multiple particle TIRM experiments are attributed to particle polydispersity (particle size variation). This is a very important result because we know that if we can maintain a relatively uniform particle size distribution, then we will see a relatively uniform stuck-particle intensity distribution. The model can then be used to select a size range of microspheres that will exhibit a more uniform distribution so as to increase the sensitivity and feasibility of multiple particle TIRM.

The technique of single particle Total Internal Reflection Microscopy (TIRM) has been used to study the scattering intensity from levitated microspheres. TIRM can be used to monitor the separation between microscopic spheres immersed in liquid (water in our case) and a surface with nm resolution. In the technique, microspheres scatter light when the evanescent waves are incident upon them. The intensity of the scattered light is directly related to the height above the surface and allows determination of the height. From the separation distance histograms, the interaction between the microsphere and interface may be characterized with a force resolution in the range of 0.01 picoNewtons. Such a system can be applied to the measurement of biomolecular interactions biomolecules attached to the microsphere and the surface. The intensity and scattering pattern of this light has been modeled using a modified Mie theory which accounts for the evanescent nature of the incident light. Diffusing Colloidal Probe Microscopy (DCPM) is an extension of the TIRM technique that simultaneously monitors multiple microsphere probes. The use of multiple probes introduces the issue of probe polydispersity. When measured at the surface, a variation in scattered light intensity of nearly one order of magnitude has been observed from a purchased microsphere sample. Thus the polydisperse collection of microspheres adds significant complexity to the scattered light signal. It is hypothesized that the dependence of the total scattered light intensity on microsphere size accounts for the scattered intensity distribution in a polydisperse microsphere sample. Understanding this variation in the scattered light with microsphere size will allow improved characterization of the microsphere/surface separation. Additionally, larger microspheres have the ability to resonantly confine light and produce spectrally narrow Whispering Gallery Modes (WGMs). It is hypothesized that WGMs may be excited in microspheres with the DCPM system. These modes may be used as a refractometric biosensor with high sensitivity to local refractive index changes on the surface of the microsphere. This research involves modeling scattered intensity distributions for polydispersed collections of microspheres based on modified Mie theory. The theoretical results are compared to experimentally obtained results and found to qualitatively explain the scattered light intensity distribution in a multiple probe DCPM system. This is an important result suggesting that microsphere size variation plays a major role in determining the distribution of scattered intensity in multiple microsphere probe systems. This work also suggests that it may be possible to excite such WGMs in a DCPM system. The introduction of WGMs would enable refractometric biosensing in such evanescent mode systems.

The technique of single particle Total Internal Reflection Microscopy (TIRM) has been used to study the scattering intensity from levitated microspheres. TIRM can be used to monitor the separation between microscopic spheres immersed in liquid (water in our case) and a surface with nm resolution. In the technique, microspheres scatter light when the evanescent waves are incident upon them. The intensity of the scattered light is directly related to the height above the surface and allows determination of the height. From the separation distance histograms, the interaction between the microsphere and interface may be characterized with a force resolution in the range of 0.01 picoNewtons. Such a system can be applied to the measurement of biomolecular interactions biomolecules attached to the microsphere and the surface. The intensity and scattering pattern of this light has been modeled using a modified Mie theory which accounts for the evanescent nature of the incident light. Diffusing Colloidal Probe Microscopy (DCPM) is an extension of the TIRM technique that simultaneously monitors multiple microsphere probes. The use of multiple probes introduces the issue of probe polydispersity. When measured at the surface, a variation in scattered light intensity of nearly one order of magnitude has been observed from a purchased microsphere sample. Thus the polydisperse collection of microspheres adds significant complexity to the scattered light signal. It is hypothesized that the dependence of the total scattered light intensity on microsphere size accounts for the scattered intensity distribution in a polydisperse microsphere sample. Understanding this variation in the scattered light with microsphere size will allow improved characterization of the microsphere/surface separation. Additionally, larger microspheres have the ability to resonantly confine light and produce spectrally narrow Whispering Gallery Modes (WGMs). It is hypothesized that WGMs may be excited in microspheres with the DCPM system. These modes may be used as a refractometric biosensor with high sensitivity to local refractive index changes on the surface of the microsphere. This research involves modeling scattered intensity distributions for polydispersed collections of microspheres based on modified Mie theory. The theoretical results are compared to experimentally obtained results and found to qualitatively explain the scattered light intensity distribution in a multiple probe DCPM system. This is an important result suggesting that microsphere size variation plays a major role in determining the distribution of scattered intensity in multiple microsphere probe systems. This work also suggests that it may be possible to excite such WGMs in a DCPM system. The introduction of WGMs would enable refractometric biosensing in such evanescent mode systems.

The technique of single particle Total Internal Reflection Microscopy (TIRM) has been used to study the scattering intensity from levitated microspheres. TIRM can be used to monitor the separation between microscopic spheres immersed in liquid (water in our case) and a surface with nm resolution. In the technique, microspheres scatter light when the evanescent waves are incident upon them. The intensity of the scattered light is directly related to the height above the surface and allows determination of the height. From the separation distance histograms, the interaction between the microsphere and interface may be characterized with a force resolution in the range of 0.01 picoNewtons. Such a system can be applied to the measurement of biomolecular interactions biomolecules attached to the microsphere and the surface. The intensity and scattering pattern of this light has been modeled using a modified Mie theory which accounts for the evanescent nature of the incident light. Diffusing Colloidal Probe Microscopy (DCPM) is an extension of the TIRM technique that simultaneously monitors multiple microsphere probes. The use of multiple probes introduces the issue of probe polydispersity. When measured at the surface, a variation in scattered light intensity of nearly one order of magnitude has been observed from a purchased microsphere sample. Thus the polydisperse collection of microspheres adds significant complexity to the scattered light signal. It is hypothesized that the dependence of the total scattered light intensity on microsphere size accounts for the scattered intensity distribution in a polydisperse microsphere sample. Understanding this variation in the scattered light with microsphere size will allow improved characterization of the microsphere/surface separation. Additionally, larger microspheres have the ability to resonantly confine light and produce spectrally narrow Whispering Gallery Modes (WGMs). It is hypothesized that WGMs may be excited in microspheres with the DCPM system. These modes may be used as a refractometric biosensor with high sensitivity to local refractive index changes on the surface of the microsphere. This research involves modeling scattered intensity distributions for polydispersed collections of microspheres based on modified Mie theory. The theoretical results are compared to experimentally obtained results and found to qualitatively explain the scattered light intensity distribution in a multiple probe DCPM system. This is an important result suggesting that microsphere size variation plays a major role in determining the distribution of scattered intensity in multiple microsphere probe systems. This work also suggests that it may be possible to excite such WGMs in a DCPM system. The introduction of WGMs would enable refractometric biosensing in such evanescent mode systems.

The technique of single particle Total Internal Reflection Microscopy (TIRM) has been used to study the scattering intensity from levitated microspheres. TIRM can be used to monitor the separation between microscopic spheres immersed in liquid (water in our case) and a surface with nm resolution. In the technique, microspheres scatter light when the evanescent waves are incident upon them. The intensity of the scattered light is directly related to the height above the surface and allows determination of the height. From the separation distance histograms, the interaction between the microsphere and interface may be characterized with a force resolution in the range of 0.01 picoNewtons. Such a system can be applied to the measurement of biomolecular interactions biomolecules attached to the microsphere and the surface. The intensity and scattering pattern of this light has been modeled using a modified Mie theory which accounts for the evanescent nature of the incident light. Diffusing Colloidal Probe Microscopy (DCPM) is an extension of the TIRM technique that simultaneously monitors multiple microsphere probes. The use of multiple probes introduces the issue of probe polydispersity. When measured at the surface, a variation in scattered light intensity of nearly one order of magnitude has been observed from a purchased microsphere sample. Thus the polydisperse collection of microspheres adds significant complexity to the scattered light signal. It is hypothesized that the dependence of the total scattered light intensity on microsphere size accounts for the scattered intensity distribution in a polydisperse microsphere sample. Understanding this variation in the scattered light with microsphere size will allow improved characterization of the microsphere/surface separation. Additionally, larger microspheres have the ability to resonantly confine light and produce spectrally narrow Whispering Gallery Modes (WGMs). It is hypothesized that WGMs may be excited in microspheres with the DCPM system. These modes may be used as a refractometric biosensor with high sensitivity to local refractive index changes on the surface of the microsphere. This research involves modeling scattered intensity distributions for polydispersed collections of microspheres based on modified Mie theory. The theoretical results are compared to experimentally obtained results and found to qualitatively explain the scattered light intensity distribution in a multiple probe DCPM system. This is an important result suggesting that microsphere size variation plays a major role in determining the distribution of scattered intensity in multiple microsphere probe systems. This work also suggests that it may be possible to excite such WGMs in a DCPM system. The introduction of WGMs would enable refractometric biosensing in such evanescent mode systems.

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In this dissertation, we describe a novel method that we call Diffusing Colloidal Probe Microscopy (DCPM), which integrates Total Internal Reflection Microscopy (TIRM) and Video Microscopy (VM) methods to monitor three dimensional trajectories in colloidal ensembles levitated above macroscopic surfaces. TIRM and VM are well established optical microscopy techniques for measuring normal and lateral colloidal excursions near macroscopic planar surfaces. The interactions between particle-particle and particle-substrate in colloidal interfacial systems are interpreted by statistical analyses from distributions of colloidal particles; dynamic properties of colloidal assembly are also determined from particle trajectories. Our studies show that DCPM is able to detect many particle-surface interactions simultaneously and provides an ensemble average measurement of particle-surface interactions on a homogeneous surface to allow direct comparison of distributed and average properties. A benefit of ensemble averaging of many particles is the diminished need for time averaging, which can produce orders of magnitude faster measurement times at higher interfacial particle concentrations. The statistical analyses (Ornstein- Zernike and three dimensional Monte Carlo analyses) are used to obtain particle-particle interactions from lateral distribution functions and to understand the role of nonuniformities in interfacial colloidal systems. An inconsistent finding is the observation of an anomalous long range particle-particle attraction and recovery of the expected DLVO particle-wall interactions for all concentrations examined. The possible influence of charge heterogeneity and particle size polydispersity on measured distribution functions is discussed in regard to inconsistent particle-wall and particle-particle potentials. In the final part of this research, the ability of DCPM is demonstrated to map potential energy landscapes on patterned surfaces by monitoring interactions between diffusing colloidal probes with Au pattern features. Absolute separation is obtained from theoretical fits to measured potential energy profiles and direct measurement by sticking silica colloids to Au surfaces via electrophoretic deposition. Initial results indicate that, as colloidal probe and pattern feature dimensions become comparable, measured potential energy profiles suffer some distortion due to the increased probability of probes interacting with surfaces at the edges of adjacent pattern features. Measurements of lateral diffusion via analysis of mean square displacements also indicated lateral diffusion coefficients in excellent agreement with rigorous theoretical predictions.