Browsing by Subject "Microscopy"
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Item Computational Digital Inline Holography for In Situ Particle Tracking and Characterization(2020-05) Mallery, KevinDigital inline holography (DIH) is a powerful single-camera 3D microscopic imaging tool that is able to digitally refocus a recorded image to reconstruct the 3D field of view. Compared to other single-camera techniques, DIH has a much larger depth of field in which objects can be seen, leading to drastically increased sampling volumes. Many particle features can be measured with DIH including size, shape, refractive index, identity, and motion. However, DIH has traditionally been limited by challenges related to the difficulty of accurately and quickly processing holographic images. In this thesis, I present technical developments focused on the digital processing of holographic images that are intended to alleviate these challenges and enable the application of DIH to new measurements. Specifically, a new approach for hologram reconstruction -- regularized holographic volume reconstruction (RIHVR) -- is introduced. This method is able to produce substantially noise-free reconstructions of particle fields. A data-driven approach to predictive particle tracking is also introduced in order to enable increased particle concentrations for particle tracking velocimetry applications. Each of these developments is validated using synthetic data and experimental demonstrations. Three applications of holographic imaging are presented to demonstrate the broad applicability of the method. The effect of temperature on the density of colonial cyanobacteria is identified by measuring the buoyant velocity and size of individual colonies. This could lead to better modelling of toxic algal blooms. Another type of algae, \emph{Dunaliella primolecta}, is useful and can be farmed for materials used in nutritional supplements, pharmaceuticals, and biodiesel. DIH is used to identify behavior signatures that could be used as indicators of optimal lipid production. This could enable optimal harvest timing leading to improved biodiesel yield. Finally, a low-cost miniature underwater holographic microscope was developed for \emph{in situ} field applications. This microscope is paired with a robotic platform to enable autonomous exploration of lakes or other aquatic environments. Despite its handheld size, the sensor is able to perform real-time particle concentration measurements using a deep neural network. The recorded images can also be used to identify the type of microorganisms found in the water.Item Examining the Role of Phospholamban Phosphorylation on Interaction with SERCA Using Fluorescence Microscopy(2018-07) Haydon, SuzanneRegulation of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) by Phospholamban (PLB) plays a crucial role in normal cardiomyocyte function through controlling the speed and extent of myocyte relaxation. The interaction between PLB and SERCA is altered in many forms of heart failure (HF), making these proteins potential targets for the treatment of HF. Both proteins have been extensively studied in vitro, where their basic structure and function were determined, and in animal models, where their role in disease was examined. However, key information connecting the in vitro experiments and animal models is needed to better understand the PLB-SERCA interaction and to design effective HF treatment strategies. In particular, we wanted to examine two conflicting in vitro models of how the PLB-SERCA interaction changes after PLB phosphorylation: the dissociation model and the structural model. In the dissociation model, phosphorylation causes PLB to dissociate from SERCA, while in the structural model, phosphorylation causes a shift in the PLB binding position along SERCA. In order to determine the correct model of PLB-SERCA interaction in live cells, we expressed cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to the N-termini of SERCA and PLB respectively, in HEK293 cells for fluorescence resonance energy transfer (FRET) microscopy experiments. We were able to use the native beta-adrenergic signaling system in the cells to control the state of PLB phosphorylation in a time-dependent manner. For the dissociation model to be true, we expected to see a significant reduction in FRET between CFP-SERCA and YFP-PLB after PLB phosphorylation. While significant increases in PLB phosphorylation were produced in the cells, FRET did not decrease. Instead, FRET increased with PLB phosphorylation at serine 16, indicating either a shorter distance between PLB and SERCA, or higher binding of PLB to SERCA. As the beta-adrenergic signal progressed through the cells, causing phosphorylation of PLB at threonine 17, FRET returned towards basal levels, but did not show the decrease below basal FRET levels that would indicate PLB dissociation from SERCA. Thus, we determined that there is a subtle change in the PLB-SERCA interaction due to PLB phosphorylation rather than a large scale dissociation. In order to differentiate changes in distance from changes in binding time-resolved (TR)-FRET experiments were required. Fluorescence lifetime imaging microscopy (FLIM) is a variant of TR-FRET that measures fluorescence decay curves with a fast-pulsed laser and photon counting board attached to a confocal microscope. These fluorescence decay curves provide more information than intensity measurements since they can be fit to multiple exponentials to test different interaction models. FLIM is a relatively new technique, thus we worked on developing appropriate experimental conditions for acquiring fluorescence decays that contained enough photons for multi-exponential fitting while still measuring individual cells. We were able to use FLIM to measure FRET values similar to those acquired on standard fluorescence microscopes and confirmed that phosphorylation of PLB did not cause dissociation from SERCA. However, further improvements to FLIM acquisition and analysis are needed for the multi-exponential fitting to provide a better model of PLB-SERCA interaction in live cells.Item A Fluorescence Assessment of the Intracellular Trafficking of the Amyloid Precursor Protein within the Secretory Pathway(2020-08) Rodriguez, KassidyAmyloid-β (Aβ) plaque formation in the brain is a major hallmark of Alzheimer’s disease (AD) and has been linked to known symptoms. According to the amyloid cascade hypothesis, Aβ is generated from the cleavage of amyloid-β precursor protein (APP) by BACE1 followed by the regulated intramembrane proteolysis (RIP) by γ-secretase. Aβ plaques are then formed through the extracellular deposition and aggregation of Aβ proteins. Current research suggests that BACE1 dimerizes in cells in a substrate-mediated manner, and that these dimers exhibit a much higher catalytic activity than their monomeric forms within acidic environments, such as endosomal compartments. It was also reported that the interaction between APP and BACE1 increased Aβ production. To date, there has been no research completed using HEK293 cells that compares the degree of co-localization of APP within specific endosomal compartments in the secretory pathway. The co-residence of APP and BACE1 in the same subcellular location would likely favor their interaction and facilitate the proteolysis of APP. Therefore, the study of their intracellular trafficking and localization should indicate where BACE1 is likely to process APP. BACE1 is an attractive therapeutic target for slowing the production of Aβ in the early stages of AD, further highlighting the importance of its study. In this contribution, we describe our efforts to examine where APP-GFP resides along the secretory pathway using fluorescently labeled Rab proteins that target distinct regions of the secretory pathway as points of reference. In addition, we observed an increase in the overall fluorescence intensity in cells co-transfected with APP-GFP and BACE1-YFP. HEK293 cells were transiently transfected with APP-GFP and either Rab5-DsRed, Rab7-DsRed, Rab11-DsRed, or BACE1-YFP fluorescent fusion proteins then imaged using widefield fluorescence and differential interference contrast (DIC) microscopy. The subcellular localization and distribution of each fluorescent fusion protein construct was determined by the colocalization of GFP and DsRed by evaluating Pearson’s correlation coefficients (PCC), Manders’ overlap coefficients (MOC), and Li’s intensity correlation quotients (ICQ). Fluorescence images were analyzed using the JACoP plug-in within FIJI/ImageJ. The results of this study provide evidence that APP-GFP has the highest degree of colocalization with Rab11-DsRed, suggesting that the APP concentration is highest within the recycling endosomes. HEK293 cells co-expressing APP-GFP and BACE1-YFP exhibited an increased fluorescence intensity compared to cells expressing only APP-GFP. These results establish baseline measurements for future cell-based studies of BACE1 with APP-GFP as its substrate.Item Microtubule Sub-Structure and its Role in Protein Binding(2018-07) Reid, TaylorMicrotubules are structural polymers that participate in a wide range of cellular functions. The microtubule binding protein EB1 localizes to the growing ends of microtubules, where it facilitates interactions of key cellular proteins with the microtubule plus-end. Recent work has demonstrated that microtubule plus-ends have open, tapered conformations, which diverge greatly from a closed tube conformation. Thus, in this work we explored whether microtubule structure could impact the binding of EB1 to microtubules. Using quantitative fluorescence and electron microcopy experiments, we found that EB1 preferentially binds structurally disrupted or open structural features of microtubules as compared to the closed microtubule lattice. In corresponding 3D single- molecule diffusion simulations, a 70-fold rise in EB1 on-rates to tapered microtubule tip structures was observed relative to a closed lattice conformation, due to a high steric hindrance barrier that impedes EB1 from binding in its four-tubulin pocket-like lattice site, with greatly increased accessibility on two-tubulin protofilament edges at tapered microtubule ends. Thus, EB1’s four-tubulin pocket-like binding site on the microtubule leads to microtubule structural recognition based on a steric-hindrance-mediated on- rate, which may allow the tapered tip structures that are typical at growing microtubule plus ends to assist in facilitating the rapid arrival of EB1 to the microtubule plus-end.Item Single cell analysis of bacterial communication and gene transfer by Enterococcus faecalis(2019-02) Erickson, RebeccaEnterococcus faecalis is a commensal member of the gastrointestinal tract of animals including humans but is also an opportunistic pathogen and a major cause of healthcare-associated infections. Its pathogenicity is thought to arise in immunocompromised people and after infection, treatment is difficult due to antibiotic resistance. E. faecalis is particularly good at transferring antibiotic resistance by mechanisms like conjugation and conjugative transfer of plasmids can occur at a high frequency without antibiotic selection. Conjugative plasmid pCF10 encodes tetracycline resistance and transfer between E. faecalis cells is facilitated by cell-to-cell communication. This signaling triggers expression of genes from pCF10 that encode for transfer machinery. The response to signaling is robust and has been extensively studied at the population level. However, it has recently become apparent that there is response variation. Understanding the mechanisms that underlie variation in response initiation is important to preventing transfer. Studies presented in this dissertation adapt fluorescence in situ Hybridization Chain Reaction (HCR) for single cell analysis of transcripts and explore questions about the pCF10 conjugation system that would not have otherwise been possible. In chapter 3, variation in the signaling response was assessed and the response was shown to be very heterogenous. When the level of signal is low, (like what might occur naturally), a minority of cells respond. Although stochasticity in the system may give rise to such heterogeneity, work in chapter 4 investigates the response impact of a few specific mechanistic players (PrgX, C, and I). Changing the levels of these components was shown to change the outcome. Lastly, single cell analysis was used in chapter 5 to assess the expression of genes required for conjugative transfer. These results show that the few responding cells commit to expression of all the genes encoding for production of the conjugation machinery. Overall, these results suggest that the pCF10 system is evolutionarily tuned for specific levels of each component and poised to have response variation for a population of cells. Thus, a small percent of cells can respond and since the majority of responding cells are able to conjugate, plasmid transfer is highly efficient. These results also exemplify how small differences in two cells can precipitate different responses in otherwise identical cells exposed to very similar conditions. Information about variation in the initiation of the signaling response required for pCF10 transfer is important to understanding the general biology of gene transfer among bacteria. In the future, this information will be important for successful design of effective interventions to the transfer of genes conferring antibiotic resistance.Item STEM standards-based reform initiatives: the impact on student learning and the curricular, instructional, and assessment practices of teachers(2013-05) Flynn, Mary LeslieNational standards in science (NRC, 1996), mathematics (NCTM 1989, 2000), and technology (NETS, 2000) have informed the direction of reform efforts to guide curriculum development, improve the instructional practices of teachers and increase student learning. STEM Standards-Based Reform Initiatives: The Impact on Student Learning and the Curricular, Instructional, and Assessment Practices of Teachers, involves a series of three studies completed around the theme of science, technology, engineering, and mathematics (STEM) standards-based reform initiatives and the impact on teacher curricular and instructional practices and student learning. Study 1, Block Scheduling and Mathematics: Enhancing Standards-Based Instruction (Flynn, Lawrenz & Shultz, 2005), is a quantitative study investigating differences in eighth-grade mathematics students' engagement in standards-based curriculum and instruction practices between block- and traditional-schedule schools. Results indicate there are few differences in curriculum and instruction based on the type of school schedule. Study 2, Building a Successful Middle School Outreach Effort: Microscopy Camp (Penn, Flynn & Johnson, 2007), focuses on the development and implementation of curriculum and instruction based on national and state standards designed to assist middle school science learners in their understanding of the atomic structure of solid crystals and the design and use of an assessment tool to monitor student understanding of the topic. Qualitative results indicate improved post-camp understanding of students' understanding of the atomic structure of solid crystals. Study 3, Integrating Technology into a Secondary Science Licensure Program: Modeling Students' Competencies to Use and Teach with Technology over the Course of the Program, is a longitudinal study modeling secondary science student teachers changes in their technology competencies over the course of their program. Results indicate teachers self-reported competencies in skill to use and preparedness to teach with technology in their science classrooms increased. Barriers to technology integration due to physical resources, knowledge and skills, and school supports are reported. Collectively, the three studies inform future research and practice in the area of STEM standards-based reform initiatives by highlighting the impact of implementation efforts across multiple disciplines and settings. Researchers and practitioners may use the research design, curriculum framework, instructional practices, assessment techniques, and results and conclusions of these studies to advance their own research and practice.Item A Study of the Regulation Mechanisms of Platelet Activation(2016-01) Finkenstaedt-Quinn, SolairePlatelets play an important role in maintaining hemostasis in the body. As they circulate through the blood stream, platelets receive signals from other cells that call them to sites of vascular damage. When platelets reach a damaged area, they go through the processes of activation and aggregation. During activation, platelets begin to change shape and release their granular contents via exocytosis. During exocytosis, a series of small molecules and proteins are released that serve to propagate the platelet activation signal and initiate wound healing. Overall, this work explores the different aspects of platelet activation using microscopy and single cell methods. Chapter One reviews the different components of the cytoskeleton and the current advances in microscopy that are being applied to study it. While light microscopy is a useful technique for studying cellular dynamics, super-resolution imaging allows for more in depth exploration of the many regulatory roles that the cytoskeleton plays in cells. Chapter Two focuses on work performed to investigate the toxicity of mesoporous silica nanoparticles on platelets. In Chapters Three and Four, the role that the cytoskeleton and plasma membrane play in the shape change dynamics of platelet activation is detailed, with a focus on using microscopy to visualize these changes. Chapter Five analyzes platelet activation to characterize fusion pore dynamics during exocytosis. Lastly, Chapter Six provides an overview of three different chemical education projects I have worked on. The first two projects involved the development and implementation of outreach events at a local community center to teach students about the scientific process and the chemistry behind climate change. The third project is in progress and aims to develop labs for use in the high school chemistry classroom that combine literature with general chemistry concepts to promote student engagement and interdisciplinary focus.Item Swimming Despite Obstacles: Bacterial Swimming as an Evolution-selected Feature(2022-08) Kamdar, ShashankIn the 1670s, Leeuwenhoek used a single-lens microscope to bring the unfamiliar microscopic world of bacteria to human attention. In this research work, we use biophysical tools of quantitative microscopy and fluid dynamics to revisit the same world of microbes and shed light on the intricate yet fascinating motion of microbes. In particular, this thesis details two fundamentally significant problems related to microbial locomotion: 1) motility of microbes in complex fluids, and 2) impact of multiflagellarity on bacterial motility. Locomotion of flagellated microorganisms is of great importance for a wide range of biological processes from disease infection, to reproduction, and to ecosystem health. Bacterial swimming in simple Newtonian fluids is well understood; however, our understanding of their motion in their natural habitats comprising of microscopic particles and polymers is still far from complete. Even after six decades of research, whether bacteria show motility enhancement in polymer solutions and what is the origin of this enhancement remain under debate. We tackled this problem from a new perspective: we studied bacterial locomotion in dilute colloidal suspensions, which do not exhibit complex rheological behaviors such as shear thinning, thickening, etc. Surprisingly, we found that all the measurable swimming features of bacteria in colloidal suspensions are quantitatively the same as those in polymer solutions. This suggests a common origin of bacterial motility enhancement in all complex fluids and challenges all the existing theories which exclusively used polymer dynamics to explain this behavior. We subsequently developed a simple hydrodynamic model considering the colloidal nature of complex fluids, which predicted bacterial motility enhancement in both colloidal suspensions and polymer solutions. We also propose a new mechanism of bacterial wobbling that shows the enhancement and also reproduced bacterial helical trajectories with large pitches—another puzzling behavior of bacterial locomotion. Thus, our study combining experiments and theory unambiguously resolved the long-standing controversy of two problems at once, i.e., the origin of bacterial motility enhancement in complex fluids and the mechanism of bacterial wobbling in Newtonian fluids. Bacterial species also show variations in their flagellar architecture and adapt two common arrangements: monotrichous or uniflagellar bacteria possess a single flagellum at the pole of their body and peritrichous bacteria grow multiple flagella over their body, which form a helical rotating bundle propelling bacterial swimming. Although the cellular features of bacteria are under strong evolutionary selective pressures, extensive studies suggest that multiflagellarity confers no noticeable benefit to bacterial motility. These findings pose a long-standing question: why does multiflagellarity emerge in bacteria given the tremendous metabolic cost of flagellar synthesis? Here, contrary to common views that seek the answer beyond the basic function of flagella in motility, we show that multiflagellarity indeed provides a significant selective advantage in bacterial motility, allowing bacteria to maintain a constant swimming speed over a wide range of body sizes. Through experiments of immense sample sizes and detailed hydrodynamic modeling and simulations, we quantitatively reveal how bacteria utilize the increasing number of flagella to regulate the flagellar motor load, which leads to faster flagellar rotational speeds balancing the higher hydrodynamic drag on the bacterial body of larger sizes. Without such an elegant mechanism, the swimming speeds of uniflagellar bacteria decrease with increasing body sizes. This stark difference between the two swimming modes provides a novel fluid dynamic insight into the crucial role of multiflagellarity in maintaining optimum motility for navigation and survival in their native habitats. Beyond, the ecological implications, results, and insights from this thesis serve as guidelines for devising artificial swimmers that efficiently navigate complex biological environments for drug delivery.