Browsing by Subject "Microscopy"
Now showing 1 - 20 of 20
- Results Per Page
- Sort Options
Item Cytoplasmic foci at the crossroads of artifactual science and biological function(2016-05) Zhao, Alice; Marcotte, Edward M.; Ellington, Andrew D; Zhang, Yan J; Appling, Dean R; Iyer, Vishwanath RDeciphering protein interaction and compartmentalization is crucial to understanding the molecular mechanisms that drive biological processes. Using various high throughput approaches, we have managed to score subcellular dynamic protein re-organization into supramolecular structures and map physical association networks to discover protein complexes on a proteome-wide level. However, the case by case studies of some of these novel structures and interactions reveal difficulties in interpreting their biological basis. This study offers insights into limits inherent in the molecular techniques used to investigate subcellular structures and protein interactions, describing a set of cautionary tales and critical analysis for deciphering cases of confounding data from orthogonal approaches. This study also offers a new experimental technique for high-throughput imaging assays with mammalian cell lines.Item Development of an NMR force microscopy probe for thin film studies(2023-08) Paster, Jeremy W.; Markert, John T.Presented here are efforts to reconfigure a nuclear magnetic resonance force microscopy (NMRFM) probe to explore thin-film samples. The motivation for this experiment, which would enlist the non-invasive and subsurface capabilities of NMRFM, was to study the conducting interface between LaAIO₃ and SrTiO₃ (LAO/STO), materials which are insulating in bulk. We propose NMRFM as a tool to detect whether there is diffusion across the interface which could explain the conducting region. We adopted the Interrupted OScillating Cyclic Adiabatic Reversal (iOSCAR) protocol to improve the signal-to-noise ratio. This thesis provides background information on nuclear magnetic resonance, NMRFM, and LAO/STO before detailing key probe transformations towards thin-film exploration with iOSCAR.Item Development of quantitative three-dimensional thermal noise imaging of biopolymer filaments(2012-05) Kochanczyk, Martin David; Florin, Ernst-Ludwig; Shubeita, George; Marder, Michael; Bengtson, Roger; Zhang, John XBiopolymer networks perform many essential functions for living cells. Most of these networks show a highly nonlinear mechanical response that is well-studied on the macroscopic scale. While much work has been done to connect the macroscopic responses of networks to microscopic parameters, such as filament stiffness, cross-linking geometry and pore size, there is a lack of experimental techniques that can measure these properties in situ. This thesis presents the development of a quantitative scanning probe imaging technique, which can explore soft matter in an aqueous environment. An optical tweezer-based microscope, called a photonic force microscope, was designed and constructed. A stability analysis method, called Power Spectrum Integration Analysis, was developed and was used to show that the photonic force microscope achieves nanometer precision in the measurement of probe position with a bandwidth of 1MHz. A novel single filament assay was developed that allowed for the isolation and probing of individual biopolymer filaments. A scanning probe technique, called thermal noise imaging, which uses the diffusive motion of an optically trapped nanoparticle as a fast, natural scanner, was used to scan microtubules grafted on one end. The resulting thermal noise images were strongly influenced by the thermally driven, transverse fluctuations of the filaments. Analytical tools, which include Brownian dynamics simulations of probe and filament, were developed to assist quantitative analysis of thermal noise images. The persistence length of individual microtubules was extracted, and the length dependence persistence length for taxol stabilized microtubules was confirmed. The transverse fluctuations of a microtubule grafted on both ends were imaged. Finally, thermal noise images of collagen filaments inside a three-dimensional collagen network were recorded, and variations of the filament diameter were extracted. This thesis establishes thermal noise imaging as a quantitative tool for studying soft material on the nanometer scale, as well as paves the way for investigating force distributions inside biopolymer networks.Item The development of Raman imaging microscopy to visualize drug actions in living cells(2001) Ling, Jian; Bovik, Alan C. (Alan Conrad), 1958-Direct Raman imaging techniques are developed and applied to study the drug distribution in single living cells. From the drug distribution, its mechanism and efficacy can be evaluated. The advantages of Raman imaging are that the Raman signals are inherent to the drug, thus no external dyes, markers or labels are required during the imaging. This makes the sample preparation much simple for the experiment. At the same time, the mechanism of action of the drug is minimally disturbed during imaging. The major challenge in Raman imaging is the weak nature of the Raman signal. In this study, the quality and sensitivity of a Raman system was improved. A model was developed to describe the degradation of Raman signals by several processes: non-uniform illumination of the excitation laser, distortion by the microscope system, and the influence of additive signal-dependent Gaussian noise that arises during imaging. Using this model, special-purpose image-processing algorithms were developed to restore the Raman images. The general Raman imaging and data analysis techniques were then applied to the visualization of drug in living cells. Taxol, an important anticancer agent whose mechanisms at the cellular level have been well studied, was used to evaluate the capabilities of direct Raman imaging. Raman images were obtained from a MDA-435 cancer cell before, during, and after the drug treatment. The results clearly show how the Taxol distribution changes with time in a living tumor cell. It was also found that Taxol does not enter the cell nucleus, but is more concentrated around the cell centrosome and near the cell membrane. This finding is explained by the binding characteristics of the Taxol and its molecular target ñ the microtubules. This result demonstrated the feasibility of using direct Raman imaging to study the distribution of anticancer agents in single living cells. Direct Raman imaging can also be extended to study drug uptake, resistance, and intracellular pharmacokinetics. I believe Raman imaging will not only provide a cost-effective tool for the study of drug mechanisms at the cellular level, but also be a general molecular imaging technique to be used in many applications.Item Effect of rigid crystals on the experimental sintering of rhyolitic ash under shallow conduit conditions(2022-12-01) Blandon, Rachel; Gardner, James Edward, 1963-; Llewellin, Edward; Ketcham, RichardWe present an experimental investigation of surface tension-driven sintering and associated densification of crystal-bearing rhyolitic ash under shallow volcanic conduit conditions. Suites of isobaric (40 MPa) experiments were held at a constant temperature of 675 to 850 °C for durations of 30 minutes to 9 hours. A 50/50 mixture of rhyolitic glass powder (<45 μm) and quartz crystals, in three distinct size distributions, were used: 250 - 500 µm, 90 - 125 µm, and 45 - 76 μm. All samples evolved similarly, from loose, cohesion-less particles to sintered particles forming a friable, agglutinated framework with an interconnected network of pores of ≥20 vol.%. All mixtures took longer to sinter and remained at a higher final porosity than crystal-free experiments performed by Gardner et al. (2018) under the same conditions. Samples sintered more slowly and remained more porous with cooler temperatures and finer crystals. Estimates for permeability indicate that these crystal-rich mixtures are as permeable as natural samples of equivalent porosity. Our experimental results suggest that solid particles inhibit the sintering process by holding porous networks open, allowing for sustained degassing. Therefore, solid particles may have important consequences for outgassing in the shallow conduit if the material is sintered together.Item Electric-field-induced second harmonic microscopy(2004) Wu, Kui; Downer, Michael Coffin.Item Fluorescence microscopy of materials with energy applications(2020-05-13) Geberth, Geoffrey Thomas; Vanden Bout, David A.; Roberts, Sean T; Baiz, Carlos; Mullins, Charles; Dodabalapur, AnanthOne of the largest drivers of modern materials research is the advancement of renewable energy, particularly solar power. This objective has seen the rise of several different materials systems, each with their own advantages and disadvantages. Before any of these alternatives to traditional inorganic semiconductors can be effectively utilized at a commercial scale, they must first be understood at the fundamental level such that they can be tuned through utilization of the structure-property relationship. This dissertation describes the use of fluorescence-based microscopy techniques to explore material systems relevant to energy production at the smallest possible levels, ranging from single molecules and aggregates to small scale surface structures in order to unravel the microscopic heterogeneities that influence photophysical performance. First, two different conjugated polymers were studied. Poly(3-(2'-methoxy-5'-octylphenyl)thiophene) (POMeOPT) aggregates were studied in bulk solution in order to probe charge transfer character in the excited state. Nonpolar solution environments led to more than hundredfold increases in the fluorescence intensity of this material, demonstrating the importance of the environment in manipulating the photophysics of conjugated polymers and illustrating the role the charge transfer state plays in the excited state. Next, a polyphenylenevinylene (PPV) based block copolymer designed for controlled folding was examined at the single molecule level with excitation polarization spectroscopy, revealing not only the robustness of the folding functionality, but also that the resulting folds spaced the chromophores far enough apart to severely limit interactions between them. Shifting to a different class of energy materials, single perylene diimide (PDI) aggregates were then formed with solvent vapor annealing (SVA) and studied with fluorescence microscopy. These experiments revealed a vast heterogeneity amongst small aggregates as well as provided strong evidence for emissive excited states with triplet character occurring even in small aggregates. The final class of material studied was a Ruddlesden-Popper phase quasi-2D organolead halide perovskite. Confocal fluorescence microscopy was utilized to image film degradation in the presence of moisture, and provided insights into the mechanism behind moisture-driven surface crystallite growth. Taken together these experiments demonstrate the power of fluorescence microscopy to advance the understanding of energy materials systems by examining small scale heterogeneity.Item High-resolution measurement of dissolved oxygen concentration in vivo using two-photon microscopy(2011-05) Estrada, Arnold Delfino; Dunn, Andrew Kenneth, 1970-; Tunnell, James; Milner, Thomas; Rylander, Grady; Ress, DavidBecause oxygen is vital to the metabolic processes of all eukaryotic cells, a detailed understanding of its transport and consumption is of great interest to researchers. Existing methods of quantifying oxygen delivery and consumption are non-ideal for in vivo measurements. They either lack the three-dimensional spatial resolution needed, are invasive and disturb the local physiology, or they rely on hemoglobin spectroscopy, which is not a direct measure of the oxygen available to cells. Consequently, many fundamental physiology research questions remain unanswered. This dissertation presents our development of a novel in vivo oxygen measurement technique that seeks to address the shortcomings of existing methods. Specifically, we have combined two-photon microscopy with phosphorescence quenching oximetry to produce a system that is capable of performing depth-resolved, high-resolution dissolved oxygen concentration (PO2) measurements. Furthermore, the new technique allows for simultaneous visualization of the micro-vasculature and measurement of blood velocity. We demonstrate the technique by quantifying PO2 in rodent cortical vasculature under normal and pathophysiologic conditions. We also demonstrate the technique’s usefulness in examining the changes in oxygen transport that result from acute focal ischemia in rodent animal models.Item Indirect selective laser sintering of ceramics(2021-12-08) Sassaman, Douglas Maxwell; Beaman, Joseph J.; Kovar, Desiderio; Seepersad, Carolyn; Ide, MatthewCeramics with intricate geometries are useful in a wide range of technical applications, but current manufacturing techniques limit the geometric complexity. Additive manufacturing (AM) is a viable approach to surmount these limitations and produce complex tailored structures. However, ceramic materials are not easily processed with AM technologies because of their high melting temperature and sensitivity to thermal shock. Because of this, an indirect approach is taken where a transient binder is added to effectively glues the ceramic particles together during shaping. Selective Laser Sintering (SLS) is capable of producing small complex polymer features, but how this capability translates to indirect SLS of ceramics is an open question. This dissertation aims to systematically investigate the limitations of using indirect SLS to produce intricate ceramic geometries. The investigation is first approached from a phenomenological perspective, where a variety of geometries are manufactured using indirect SLS and then compared to similar polymer parts produced with direct SLS. The geometry comparison is paired with a mechanistic study of particle-scale interactions using in-situ microscopy, and an analytical model is developed to describe these observations. Guidelines for the geometries possible with polymer SLS turn out to be a good starting place for the design and manufacture of ceramic geometries using indirect SLS. However, indirect SLS is further limited by the heterogeneity of the blended powder. In-situ microscopy shows that, in mixed polymer/ceramic powders, the binding mechanisms are different at different laser scan speeds and also different from polymer SLS or selective laser melting of metals. A permeation model is used to describe the binding mechanisms specific to indirect SLS and correctly predicted all of the trends observed in experiments. The settings (laser power and scan speed) that produced parts strong enough to be removed from the SLS machine were predicted by the model to produce permeation distances large enough for particle bonding. Conversely, settings which resulted in failed parts during the experiments were predicted by the model to not permeate far enough for particle bondingItem Line excitation array detection microscopy(2019-07-16) Martin, Christopher Michael; Ben-Yakar, Adela; Dunn, Andrew; Tunnell, James; Nauhaus, IanHigh-speed, volumetric fluorescence and two-photon imaging systems are a necessity for studying biological systems. To screen drug compounds using small animal models, hundreds of animals must be imaged in seconds. To discover how the brain gives rise to behavior and disorders, neural circuits must be monitored at the millisecond timescales of action potentials. This dissertation develops a high-speed scanned light-sheet imaging method, called line excitation array detection (LEAD) microscopy, to meet the needs of such applications. LEAD microscopy combines fast line-scanning with an acousto-optic deflector and fast, sensitive imaging with a linear photomultiplier tube array to reach high frame rates. LEAD microscopy was first implemented as a high-throughput fluorescence cytometer for the small nematode Caenorhabditis elegans. An acousto-optic deflector scans an excitation line across a field-of-view covering the C. elegans cross-section at 800,000 Hz. Fourteen elements of a linear photomultiplier tube array image the line as it scans to generate 66×14 pixel frames at 800,000 frames per second. Individual animals flowing through the light-sheet at 1 meter per second were imaged in 1 millisecond, and populations of hundreds were imaged in seconds. The cytometer has an average resolution of ~3.5 μm in each dimension, with a signal-to-noise ratio over 20 when imaging C. elegans. The system’s potential for high-throughput drug screening was demonstrated by imaging thousands of polyglutamine-mediated aggregation model C. elegans. Statistically-significant phenotypic differences between healthy and unhealthy animals after drug treatment were identified in under a second. Future LEAD cytometers can reach higher resolutions and field-of-views without sacrificing frame rate. LEAD microscopy was then developed for two-photon imaging of brain activity. A galvanometric mirror scans a ~1×22 μm² line across the field-of-view at 1,300 Hz, and the photomultiplier tube array images the line as it scans to reach 2,600 frames per second. Fluorescent beads are imaged with a lateral resolution of 1 – 2 μm. The incorporation of temporal focusing enables an axial resolution of ~5 μm, and reduces out-of-focus fluorescence. A higher-speed two-photon LEAD microscope with acousto-optic scanning at up to 200,000 frames per second and 400 volumes per second is designed for high-speed imaging of action potentialsItem Measurement of transient transport of hyperosmotic agents across cell membranes and resulting optical clearing using differential phase contrast optical coherence microscopy(2005) Rylander, Christopher Grady, 1978-; Diller, K. R. (Kenneth R.)The response of tissue to hyper-osmotic agents is a reduction in light scattering and corresponding increase in optical clarity. “Tissue optical clearing” permits delivery of near-collimated light deeper into tissue potentially improving the capabilities of optical diagnostic and therapeutic applications. The overall objective of the proposed research is to characterize the mass transport of hyper-osmotic agents across cell membranes and the resulting optical clearing. To accomplish this task, a differential phase contrast optical coherence microscope (DPC-OCM) is configured to permit quantitative spaciotemporal optical path length (OPL) imaging of biological cell specimens. The first application of DPC-OCM is analyzing the intracellular dry mass of individual biological cells. Differences between normal and cancerous cell dry mass are investigated. Populations of normal and cancerous human dermal fibroblast cells and human prostate cells demonstrate a statistically significant difference in mean dry mass and mean en face area. Linear discriminant analysis yields a maximum of 79% accurate classification. The second application of DPC-OCM is use as a novel technique for determining cell membrane permeability parameters due to an osmotic chemical stimulus. Glycerol, a hyperosmotic agent, is perfused across an adherent layer of human keratinocytes, and the dynamic osmotic response of individual cells is imaged with DPC-OCM. A novel optical path length (OPL) mass transport model is devised relating chemical concentration to intrinsic refractive index and OPL. Hydraulic conductivity and solute permeability are determined by fitting the OPL mass transport model to transient OPL data collected with DPC-OCM. The final study investigates the mechanisms of optical clearing of cellular and collagenous tissue using hyperosmotic agents and evaporation. OCT and photographic images quantify optical scattering reduction between native and dehydrated tissue states. Air-drying optically clears tissue as effectively as the most successful hyperosmotic agent, glycerol. Tissue ultrastructural alterations due to dehydration are investigated using transmission electron microscopy. The Rayleigh-Gans model is used to simulate light scattering effects due to tissue ultrastructural alterations and measured refractive index excursion using DPC-OCM.Item Methods development and measurements for understanding morphological effects on electronic and optical properties in solution processable photovoltaic materials(2012-12) Ostrowski, David Paul; Vanden Bout, David A.; Rossky, Peter J; Holliday, Bradley J; Korgel, Brian A; Dodabalapur, Ananth JThe effects of morphology on electronic and optical properties in solution processable photovoltaic (PV) materials have been studied through two different approaches. One approach, scanning photocurrent (PC) and photoluminescence (PL) microscopy, involved mapping PC generation and PL in functional PV devices on the length scale of around 250-500 nm. Additionally, local diode characteristics were studied from regions of interest in the PV through local voltage-dependent photocurrent (LVPC) measurements. In a PV made from a Copper Indium Gallium Selenide (CIGS) nanocrystal (NC) "ink", two morphological features were found to cause the spatial heterogeneity in PC generation. Cadmium Sulfide (CdS) aggregates lowered PC generation by blocking incident light to the photoactive layer, and cracks in the CIGS-NC film enhanced PC generation through improved charge carrier extraction. LVPC measurements showed all regions to have similar diode characteristics with the main difference being the PC generated at zero bias voltage. For another PV made from a donor/acceptor blend of poly(9,9-dioctylfluorene-co-bis-N,N-(4-butylphenyl)-bis-N,Nphenyl- 1,4-phenylenediamine (PFB) and poly(9,9-dioctylfluorene-co-benzothiadiazole)(F8BT), two incident laser wavelengths were used to selectively illuminate only one or both polymers. The results showed that when F8BT is illuminated, the PFB-rich regions produced the most PC and when both polymers are illuminated (but mostly PFB), the F8BT-rich regions produce the most PC; showing PC generation is more affective when less absorber material is present in the morphology. The other approach to study morphological effects on PV properties was to fabricate particles that mimicked morphological variations known to occur in solution-processable PVs. Through solution processing of an oligothiophene molecule, a range of weakly coupled H-aggregate particles were made. These particles, identifiable by shape, were shown to have a varying degree of energetic disorder (as gauged by the 0-0 vibronic band intensity in the emission spectrum), despite all particles showing a similarly high degree of molecular order from fluorescence dichroism (FD) measurements. A trend was observed correlating a decrease in energetic disorder with an increase in the local contact potential (LCP) difference as measured with Kelvin probe force microscopy (KPFM). The LCP difference was found to range by 70 mV between particles of moderate to low energetic disorder.Item Nonlinear microscopy methods for imaging and particle tracking in thick biological specimen(2017-05) Perillo, Evan Paul; Dunn, Andrew Kenneth, 1970-; Yeh, Tim H. C.; Milner, Thomas E; Zheng, YuebingOptical microscopy techniques such as single-particle tracking and high-resolution (<500 nm) imaging are critical tools for the advancement of biological research. However most high-resolution optical techniques utilize a camera-based or confocal-based detection scheme, which limits the working distance into samples to approximately 10 μm due to light scattering. Nonlinear excitation methods, such as two- and three-photon microscopy, have enabled imaging in thick and scattering samples due to their longer excitation wavelengths and absence of spatial filtering. However, nonlinear excitation is rarely utilized for single-particle tracking as it traditionally offers slightly worse resolution than the aforementioned methods. This dissertation presents the progress made towards adapting nonlinear excitation for high-resolution biological study at scales ranging from single-molecules up to entire tissues. We describe a novel single-particle tracking microscope based upon multiplexed nonlinear illumination, coined TSUNAMI. Single-particle tracking with nanometric resolution using TSUNAMI is demonstrated in live cells and spheroid tumor models to unprecedented depths of 200 μm. Several new long wavelength excitation laser sources are detailed which provide superior image penetration depth compared with traditional sources. Furthermore, we detail a newly discovered form of nonlinear excitation, based upon a two-color, three-photon absorption process, and discuss potential benefits of this new excitation regime. The systems and methods developed in this work will provide life scientists with a powerful toolset for the future of biological research.Item Optical coherence tomography for retinal diagnostics(2013-08) Yin, Biwei; Milner, Thomas E.; Rylander, H. Grady (Henry Grady), 1948-Optical Coherence Tomography (OCT) is a non-invasive three-dimensional imaging technique. OCT synthesizes a cross-sectional image from a series of lateral adjacent depth scans, and with a two-dimensional scanning scheme, three-dimensional intensity image of sample can be constructed. Due to its non-invasive capability, OCT has been widely applied in ophthalmology, cardiology and dermatology; and in addition to three-dimensional intensity image construction, various functional OCT imaging techniques have been developed for clinical application. My research is focused on developing functional OCT systems for application in ophthalmology, including polarization-sensitive optical coherence tomography (PS-OCT) for retinal nerve fiber layer (RNFL) birefringence measurement and dual-wavelength photothermal optical coherence tomography (DWP-OCT) for microvasculature blood oxygen saturation (SO2) measurement. In the study, a single-mode-fiber based polarization-sensitive swept-source OCT (PS-SS-OCT) with polarization modulator, polarization-sensitive bulk-optics balanced detection module is constructed and polarization processing methods based on Stokes vectors are applied to determine birefringence. PS-OCT is able to provide human subject's RNFL thickness, phase retardation, and birefringence information. Degradation in the degree of polarization (DOP) along depth is investigated and its difference between four quadrants of RNFL (superior, temporal, inferior and nasal) indicates the structural property difference. DWP-OCT is a novel functional OCT system consisting of a phase-sensitive optical coherence tomography system (PhS-OCT) and two photothermal excitation lasers. PhS-OCT is based on a swept-source laser operating in the 1060 nm wavelength range; the two photothermal excitation lasers with wavelength 770 nm and 800 nm are intensity modulated at different frequencies. PhS-OCT probe beam and two photothermal excitation beams are combined and incident on the sample, optical pathlength (op) change on the sample introduced by two photothermal excitation beams are measured and used for blood SO2 estimation. A polarization microscope is proposed for future study. The polarization microscope is an imaging technique providing molecular structure and orientation based on probe light's polarization state information. The polarization microscope uses a wavelength tunable light source, and can achieve any incident polarization state by a retarder-rotator combination. Specimen's birefringence can be determined based on the changing of detected light amplitude.Item Photoacoustic microscopy of nanoparticles in cells and tissues(2013-05) Cook, Jason Ray; Emelianov, Stanislav Y.; Pearce, John A., 1946-Molecular photoacoustic imaging is an exciting new field that promises to visualize molecular indicators of disease. The objective of this dissertation is to progress molecular imaging by providing a photoacoustic microscopy platform to better validate in vivo molecular photoacoustic imaging, diagnose disease, and study fundamental photoacoustic processes. Initially, a custom photoacoustic microscope was developed to provide high-sensitivity and high-resolution of both endogenous and exogenous contrast agents in thin cell or tissue samples. After characterization, the photoacoustc microscope was first used to image the hemoglobin distribution in the spleen and liver. The photoacoustic microscope was then used to image nanoparticles in injured and diseased cell and tissues samples. These images can be used for in vivo photoacoustic image validation or, independently, as a diagnostic tool for disease. To enhance the utility of photoacoustic microscopy, a quantitation technique was developed for nanoparticles in cells and tissues. Quantitative photoacoustic imaging has the potential to replace mass spectrometry and histology for a wide array of molecular imaging and targeting studies. Finally, photoacoustic microscopy was used to study the nonlinear dependence of the photoacoustic pressure with laser fluence of nanoparticle-loaded cells. New discoveries about the nonlinear dependence with nanoparticle concentration and cell type are presented. These new discoveries may provide the framework for a new type of photoacoustic imaging with contrast that is cell-type specific. Overall, the work described in this dissertation can be used to improve diagnosis and accelerate clinical translation of new and emerging molecular imaging techniques.Item Scientific analysis of Thalassia testudinum leaves(2022-08-12) Wilson, Nathan Gauntlett; Wilson, Preston S.; Ballard, Megan SSeagrass meadows are an important element of coastal ecosystems and they perform numerous environmental functions. Because of their importance, the ability to properly monitor the health of the seagrass meadows is imperative. Seagrass can greatly affect acoustic propagation and therefore acoustics can be used for remotely sensing the health of the meadow. To do so, an accurate model of sound propagation within seagrass is required. Knowledge of the volume of gas contained within seagrass leaves is a requisite part of the process. The first two chapters of this report describe the microscopic imaging, and image analysis, of seagrass leaves that were used in associated laboratory acoustic measurements. The last chapter of this report describes the preparation of an acoustic measurement apparatus, a one-dimensional resonator tube, that was used to measure the effective sound speed of seagrass tissue in water. The results themselves and discussion of the measurements are outside the scope of the present report but citations that direct the reader to those results are provided.Item Secondary functionalization of passivated Si(111) : pathways to stable photoelectrode systems(2021-05-06) Gurrentz, Joseph Martin; Rose, Michael J., Ph. D.; Roberts, Sean T; Milliron, Delia J; Ekerdt, John GSilicon surfaces functionalized with electrochemically-active metal complexes and clusters are an important family of functional interfaces. These systems have shown promise for solar energy conversion and storage, molecular electronics, and biological and chemical sensing applications. However, surface stability is a limiting factor that has persistently challenged the broad implementation and in-depth study of photoelectrochemistry at chemically-modified Si surfaces. The work presented herein demonstrates that secondary functionalization of passivated Si(111) surfaces is a versatile means to generate stable Si-based photoelectrodes comprised of surface-tethered metal-containing redox species. Organic mixed monolayers and metal oxide overlayers were used as platforms for the immobilization of a macro-chelated Ni(II)-bis-diphosphine complex and heteropolytungstate clusters on oxide-resistant Si(111). Rigorous physical and (photo)electrochemical analyses were used to probe analyte surface coverages, asses interfacial electron transfer kinetics, and indicate the extent of band bending in each sample. Thus, the high stabilities of these samples’ surfaces were revealed, which enabled deconvolution of kinetic and thermodynamic structure-function relationships that govern photoelectrochemical outcomes of these systems. These studies revealed the importance of band-edge modulation as a primary design consideration in the development of optimized chemically-modified photoelectrodes.Item Study of applications of second harmonic generation(2011-05) Prem, Adrienne Marie; Downer, Michael Coffin; Sitz, Greg O.Two applications of second harmonic generation (SHG), a nonlinear optical technique, are studied. First, Fresnel factors are used with a bond model to describe SHG from vicinal silicon at five incidence angles: 7.5°, 22°, 30°, 45°, and 52°. Second, a prototype apparatus for applying SHG to enhance imaging capabilities of optical coherence tomography, a microscopy technique used in many biological fields, is briefly described.Item Study of two-photon Line Excitation Array Detection microscopy(2023-04-21) Murphy, Samuel Alexander; Ben-Yakar, AdelaThe functional meaning associated with neuronal activity in the mammalian brain is an active area of research limited by the available microscope instrumentation. Exploring this domain of neuroscience necessitates high-speed 3D imaging operating over 1 kHz volumetric scan rates with sub-cellular resolution, as action potentials propagate on sub-millisecond time scales. Monitoring these signals requires in vivo experimentation, so additional care must be taken to avoid invasive methods that may damage sample tissue to live animal subjects. Multi-photon imaging provides an opportunity for non-invasive microscopy with optical sectioning while simultaneously deeply penetrating brain tissue. However, current multi-photon microscopy methods are limited to 10-100 Hz volumetric imaging rates. This thesis explores and expands upon a potential high-speed 2-photon imaging technology, 2-photon Line Excitation Array Detection (2p-LEAD) microscopy. 2p-LEAD combines line scanning with detection via a multi-channel photomultiplier tube (PMT) array, with the potential to operate at 125 kHz frame rates. In the experimental prototype outlined in this thesis, a 1035 nm excitation line of 2.4 µm x 259 µm (1/e2 beam intensity diameter) is scanned at the focal plane. The resulting fluorescence is collected by a 16-channel linear PMT array. With a fast-scanning galvanometric mirror, we scan the excitation line at 3,000 FPS, generating a 170 µm x 75 µm fluorescence FOV imaged to a 16 x 320 pixel frame. Temporal focusing was implemented to improve optical sectioning and signal-to-noise ratio (SNR), by reducing the out of focus fluorescent signal. This reduction was achieved by dispersing the pulse-width from 300 fs at the focus to multiple picoseconds. 0.5-2 µm fluorescent polystyrene beads were imaged to characterize the system resolution of 1-5.3 µm laterally. Thus this research lays the groundwork for 2p-LEAD imaging at 125 kHz, with an acousto-optic deflector replacing the galvo-mirror as the primary scanning element, for high-speed neuronal imaging.Item Using correlated super-resolution optical and structural studies to investigate surface-enhanced Raman scattering on silver & gold nanoparticles(2015-05) Weber, Maggie Leona; Brodbelt, Jennifer S.; Willets, Katherine A.; Hoffman, David W; Korgel, Brian A; Roberts, Sean T; Anslyn, Aric V; Holcombe, James ASurface-enhanced Raman scattering (SERS) from organic molecules is used to study how geometry affects plasmonic enhancement in noble metal nanoparticles using super-resolution analysis techniques coupled with scanning electron microscope (SEM) imaging. We demonstrate that super-resolution analysis can track emission from Raman-active molecules on nanoaggregate surfaces with resolution typically better than 5 nm, a significant improvement over the diffraction limit. This optical analysis technique is used to identify active junction regions, known as hot spots, in correlated SEM structural images of aggregated particles based on shape, size, and angular position of SERS emission. This correlated analysis is further extended to study the mechanism of silver nanoparticle luminescence and its positional relationship to SERS emission. These studies support the hypothesis that silver luminescence is enhanced via a different mechanism than SERS. Discrete dipole approximation calculations agree with experimental results, indicating that SERS emission is a product of local plasmonic enhancement in specific junction regions between nanoparticles whereas silver luminescence is dependent upon nanoaggregate geometry and the collective plasmon modes within a structure. These theoretical calculations of luminescence centroid location can assist with SERS-active junction region assignment in higher order aggregates (e.g. trimers, tetramers, etc.) Finally, correlated optical and structural studies are used to develop understanding of site-specific electrochemical potentials on silver and gold nanoparticle aggregates using a redox active Raman reporter molecule. We demonstrate that there is a weak correlation between emission centroid location and bulk sample potential. Silver colloid studies demonstrate variability in modulation behavior and are highly unpredictable, whereas gold colloids with the redox molecule covalently tethered to their surface via a gold-thiol bond show more reproducible modulation. Furthermore, these tethered studies demonstrate geometrical agreement between site-specific emission at negative potentials and junction regions in nanoaggregate SEM images indicating that plasmonic enhancement may affect local nanoparticle surface potentials.