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.

Spatial and temporal tracking of passively diffusing functionalized colloids continues to be an improving and auspicious approach to measuring weak specific and non-specific biomolecular interactions. Evidence of this is given by the recent increase in published studies involving the development and implementation of these methods. The primary aim of the work presented in this dissertation was to modify and optimize video microscopy (VM) and total internal reflection microscopy (TIRM) methods to permit the collection of equilibrium binding and sampling data from interaction of surface-immobilized biomolecules. Supported lipid bilayers were utilized as model systems for functionalizing colloid and wall surfaces. Preliminary results measuring calcium-specific protein-protein interactions between surface immobilized cadherin fragments demonstrate the potential utility of this experimental system and these methods. Additionally, quantum dot-modified colloids were synthesized and evanescent wave-excited luminescence from these particles was used to construct potential energy profiles. Results from this work demonstrate that colloids can be used as ultra-sensitive probes of equilibrium interactions between biomolecules, and specialized probes, such as those modified with quantum dots, could be used in a spectral multiplexing mode to simultaneously monitor multiple interactions.

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.

This research presents a detection scheme for analyzing the temperature distribution produced by the Joule heating effect nearby the channel wall in a microfluidic chip utilizing a temperature-dependent fluorescence dye. An advanced optical microscope system¡Xtotal internal reflection fluorescence microscope (TIRFM) is used for measuring the temperature distribution on the inner channel wall at the point of electroosmotic flow in an electrokinetically driven microfluidic chip. In order to meet the short working distance of the objective-type TIRFM, microscope cover glass are used to fabricate the microfluidic chips. The short fluorescence excitation depth from a TIRFM makes the intensity information obtained is not sensitive to the channel depth variation which ususally biases the measured results while using conventional epi-fluorescence microscope (Epi-FM). Therefore, a TIRFM can precisely describe the temperature profile of the distance within hundreds of nanometer of the channel wall where consists of the Stern layer and the diffusion layer for an electrokinetic microfluidic system. In order to investigate the temperature distribution produced by the Joule heating effect for electrokinetically driven microchips, this study not only measures the temperature on the microchannel wall by the proposed TIRFM but also measures the temperature inside the microchannel by an Epi-FM. In addition, this research presents a method to reduce the Joule heating effect and enhance the separation efficiency of DNA biosamples in a chip-based capillary electrophoresis (CE) system utilizing pulse DC electric fields. Since the average power consumption is reduced by the pulse electric fields, the Joule heating effect can be significantly reduced. Results indicate the proposed TIRFM method provides higher measurement sensitivity over the Epi-FM method. Significant temperature difference along the channel depth measured by TIRFM and Epi-FM is experimentally observed. In addition, the measured wall temperature distributions can be the boundary conditions for numerical investigation into the Joule heating effect. The proposed method gives a precise temperature profile of microfluidic channels and shows the substantial impact on developing a simulation model for precisely predicting the Joule heating effect in microfluidic chips. Moreover, in the research of reducing the Joule heating effect and enhancing the separation efficiency in a chip-based CE system utilizing pulse electric fields, the experimental and numerical investigations commence by separating a mixed sample comprising two fluoresceins with virtually identical physical properties. The separation level is approximately 2.1 times higher than that achieved using a conventional DC electric field. The performance of the proposed method is further evaluated by separating a DNA sample of Hae III digested £XX¡V174 ladder. Results indicate the separation level of the two neighboring peaks of 5a (271 bp) and 5b (281 bp) in the DNA ladder is as high as 120% which is difficult to be achieved using a conventional CE scheme. The improved separation performance is attributed to a lower Joule heating effect as a result of a lower average power input and the opportunity for heat dissipation during the zero-voltage stage of the pulse cycle. Overall, the results demonstrate a simple and low-cost technique for achieving a high separation performance in CE microchips.

Semiconductor ring laser diodes (SLD) have been receiving attention for their potential use as source in photonic integrated circuits. Advantages of a ring laser include ease of integration because of no need for cleaved facets and they can be made very compact by folding their cavity . Ring laser have a unique feature, clockwise and counter- clockwise, in their lasing modes. If unidirectional traveling-wave oscillation can be achieved, spatial hole burning effects seen in Fabry-Perot and distributed feedback lasers can be avoided. In this work, the unidirectional oscillation is accomplished by controlling the taper shape structure. The whole laser cavity is formed using four reflection mirrors (TIR) and an output coupler passive waveguide. According to the Beam propagation Method (BPM) simulation, we find that the clockwise and counterclockwise oscillations have different behavior under various taper shape , indicating bidirectional oscillation can be eliminated. Moreover, bending loss¡Bmode transformation and optical gain are all included in calculation model. The waveguide is fabricated in the following steps: (1) ion implantation to get electrical isolation (2) selectively wet etching to form waveguide ridge (3) evaporation n- and p- electrode (4)spatter with Si3N4(5) planarization (6) evaporation microwave transmission line.

Fluorescence microscopy allows for spatial and temporal resolution of systems which are inherently fluorescent or which can be selectively labeled with fluorescent molecules. Temporal resolution is crucial for imaging real time processes in living samples. A common problem in fluorescence microscopy of biological samples is autofluorescence, fluorescence inherent to the system, which interferes with detection of fluorescence of interest by decreasing the signal to noise ratio. Two current methods for improved imaging against autofluorescence are two-photon excitation and total internal reflection microscopy. Two-photon excitation occurs when two longer wavelength photons are absorbed quasi-simultaneously by a single fluorophore. For this to take place there must be a photon density on the order of 1030 photons/(cm2)(s), which is achieved through use of a femtosecond pulsed laser and a high magnification microscope objective. Two-photon excitation then only occurs at the focal spot, significantly reducing the focal volume and therefore background autofluorescence. The second method, total internal reflection, is based on evanescent wave excitation, which decreases exponentially in intensity away from the imaging surface. This allows for excitation of a thin (~200 nm) slice of a sample. Since only a narrow region of interest is excited, an optical slice can be imaged, decreasing excitation of out-of-focus autofluorescence, and increasing the signal to noise ratio. By coupling total internal reflection with two-photon excitation, an entire cell can be imaged while still maintaining the use of lower energy photons to irradiate the sample and achieve two-photon excitation along the length traveled by the evanescent wave. This system allows for more sensitive detection of fluorescence of interest from biological systems as a result of a significant decrease in excitation volume and therefore a decrease in autofluorescence signal. In the two-photon total internal reflection microscopy setup detailed in this work, an excitation area of 20 μm by 30 μm is achieved, and used to image FITC-stained actin filaments in BS-C-1 cells

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Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

The Min protein system regulates cell division in the bacterium Escherichia coli. The protein MinD undergoes a pole-to-pole oscillation, antagonizing formation of the division septum at the cell poles, thereby confining the septum formation to the mid-cell. The MinD oscillation period is 40 s at room temperature in healthy cells, but has been shown to be sensitive to stress on the cell. By fluorescently labeling MinD with green fluorescent protein (GFP), we are able to measure the MinD oscillation period as an in situ metric of cell viability using high resolution total internal reflection fluorescence (TIRF) microscopy. We have made several improvements to the method by which we measure and analyse the MinD oscillation period. A microscopy flow cell was designed and constructed and it provides temperature control and stability to a precision of 0.05 °C in addition to allowing controlled addition of bacterial cells and reagents of interest to the imaging region of the flow cell. This flow cell enabled us to make a precise measurement of the temperature dependence of the MinD oscillation period, for which we observed an Arrhenius dependence with an activation energy of 11.8 kcal/mol. We developed a centroid-tracking method, performed in a custom MATLAB program, to extract the values of the MinD oscillation periods from our time series of TIRF microscopy images. We measured the effect on the MinD oscillation period of exposure to the cationic antimicrobial peptide polymyxin B (PMB) and the related compound polymyxin B nonapeptide (PMBN), which does not have antimicrobial activity. Exposure to PMB resulted in a 60% increase in the average MinD oscillation period tau, whereas exposure to PMBN resulted in an 20% decrease in tau. After exposure to PMB and PMBN, we measured the Arrhenius temperature dependence of the MinD temperature dependence and calculated the associated activation energy Ea. We found that exposure to PMB resulted in a 40% increase in Ea, whereas exposure to PMBN did not significantly change the value of Ea. These results indicate that careful measurements of the MinD oscillation can yield information that can be helpful in evaluating the mechanism of action of antimicrobial compounds. / Natural Sciences and Engineering Research Council

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.