Bioimaging and Biomedical Optics Commons^™

A Functional Ultrasound Imaging (Fusi) Pipeline For Investigating Brain-Wide Hemodynamics During Torpor-Like Hypothermia, Owen J. Culver

McKelvey School of Engineering Graduate Student Theses & Dissertations

Torpor is a regulated state of reduced metabolic demand that enables animals to conserve energy under unfavorable environmental conditions. The controlled induction of a similar state, termed synthetic torpor, has potential applications in ischemic protection, organ preservation, cancer treatment, radiation protection, and long-term spaceflight. Although neuronal populations within the preoptic hypothalamus have been shown to initiate hypothermic and hypometabolic states, the distributed cerebral hemodynamic dynamics accompanying their induction and maintenance remain poorly understood. In this thesis, we developed an accessible functional ultrasound imaging (fUSI) acquisition and analysis pipeline and applied it to investigate cerebral hemodynamics during chemogenetic activation of the …

Single Fluorogens And Orientation-Localization Microscopy For Quantifying Chemical And Biomolecular Dynamics At The Nanoscale, Yiyang Chen, Yuanxin Qiu, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Many chemical systems look uniform only because ensemble measurements average over their most interesting molecules. Electron-transfer rates vary across electrode surfaces; lipid membranes contain nanodomains with distinct packing and fluidity; peptide aggregates exhibit local polymorphism; and biomolecular condensates contain transient networks of interactions that are blurred in ensemble images. A central challenge in chemical imaging is not simply to see smaller structures, but to measure chemical variables such as polarity, redox potential, and molecular confinement at the single-molecule level. In this Account, we describe how fluorogens, molecules whose brightness, blinking, spectral shifts, orientation, rotational mobility, and motion are directly shaped …

Sensing Negative-Cone Rotational Diffusion Of Dipole-Like Emitters, Yuanxin Qiu, Kaizhi A. Nie, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Fluorescence anisotropy and single-molecule orientation-localization microscopy (SMOLM) are powerful techniques that quantify the rotational diffusion of dipole-like emitters, which is important for sensing molecular interactions and chemical environments at the nanoscale. Numerous theoretical and experimental studies have thoroughly characterized single-molecule rotations even when those rotations are much faster than the detector integration time. Here, we extend the theory of measuring rotational diffusion to situations where a single dipole rotates uniformly everywhere outside of an isotropic cone of a certain size, termed a negative cone. This scenario corresponds to negative fluorescence anisotropy 𝑟 and has been observed in emitters exhibiting strong …

Single-Molecule Orientation And Localization Microscopy, Sophie Brasselet, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Single-molecule localization microscopy (SMLM) offers enhanced spatial resolution in optical microscopy, providing detailed insights into the spatial organization of proteins in cells at the nanoscale. Over the past decade, SMLM has progressively incorporated the capability to retrieve the orientations of single molecules using their polarized dipolar emission pattern. Here we explore recent advancements in single-molecule orientation and localization microscopy (SMOLM), which yields super-resolved images of molecular three-dimensional (3D) orientations, wobble and 3D positions. This advancement opens possibilities to explore the nanoscale organization and conformation of biological molecules as well as to monitor and design local 3D optical fields in nanophotonics. …

Development Of A Light-Based Platform To Assess Cervical Mucus Barrier Function And Physical Mucus Properties, Elizabeth J. Buzbee

McKelvey School of Engineering Graduate Student Theses & Dissertations

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide, yet current clinical tools for predicting its risk lack specificity and reliability. Cervical mucus plays a crucial role in maintaining pregnancy by forming a barrier that protects the uterus against infection. This study investigates the use of laser speckle rheology as a noninvasive technique to assess cervical mucus biomechanics. Synthetic cervical mucus was fabricated at varying hydration levels and tested using a conventional rheometer that confirmed that the mechanical properties of the synthetic mucus samples were representative of physiological cervical mucus. These results were compared to the complex …

Automated Beam Stitching And Segmentation Procedure For Space Division Multiplexing Optical Coherence Tomography Angiography, Andrew J. Song

McKelvey School of Engineering Graduate Student Theses & Dissertations

Optical Coherence Tomography Angiography (OCTA) has revolutionized ophthalmic imaging and its capability to produce high-resolution 3D maps of the retinal microvasculature is instrumental in diagnosing retinovascular diseases such as diabetic retinopathy and age-related macular degeneration; However, the existing OCTA devices often suffer from slow acquisition speed limiting the field-of-view (FOV) in the clinic. Space Division Multiplexing OCTA (SDM-OCTA) address these limitations by acquiring multiple beams simultaneously, achieving manyfold faster acquisition speeds than single beam OCTA systems. But as each beam contains only part of the image, SDM-OCTA requires additional processing steps to produce coherent wide-field images. Though manual stitching and …

Painting Rich Six-Dimensional Pictures Using Polarized Fluorescence Microscopy, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

No abstract provided.

Single-Molecule Orientation Imaging Reveals The Nano-Architecture Of Amyloid Fibrils Undergoing Growth And Decay, Brian Sun, Tianben Ding, Weiyan Zhou, Tara S. Porter, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Amyloid-beta (Aβ42) aggregates are characteristic Alzheimer’s disease signatures, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational “wobble” of Nile blue (NB) molecules transiently binding to Aβ42 fibrils. We correlate fibril architectures measured by SMOLM with their growth and decay over the course of 5 to 20 min visualized by single-molecule localization microscopy (SMLM). We discover that stable Aβ42 fibrils tend to be well-ordered and signified by well-aligned NB orientations and small wobble. SMOLM also shows that increasing order …

Data-Driven Insights Into Spatial Patterns And Disease Etiologies Of White Matter Hyperintensities, Sugandha Roy

McKelvey School of Engineering Graduate Student Theses & Dissertations

In this thesis, we have applied Orthogonal Projective Non-Negative Matrix Factorization (opNMF) to identify spatial patterns of white matter hyperintensities (WMH) within UK Biobank's imaging data. Our selection criteria excluded subjects with a history of neurological, mental, and specific cerebrovascular conditions, allowing us to focus on WMH patterns in a healthy aging population. We have interrogated the association of location-specific WMHs with a variety of demographic, clinical, and genetic factors. Our multivariable regression analysis evaluates the strength and nature of the associations between these factors and WMH distribution. The analysis integrates variables such as age, sex, smoking habits, medication usage …

An Attention Lstm U-Net Model For Drosophila Melanogaster Heart Tube Segmentation, Xiangping Ouyang

McKelvey School of Engineering Graduate Student Theses & Dissertations

Machine learning is commonly used in biomedical image analysis, as it allows automated image segmentation and identification that minimizes the need for tedious human involvement. Drosophila melanogaster is often used as a cardiac disease model, where optical coherence microscopy (OCM) is used to image and analyze its beating dynamics. As OCM often generates a large volume of images, automated image segmentation is necessary to quantify the heart beating efficiently. Our most recent heart segmentation model, FlyNet 2.0+, is a fully convolutional LSTM U-Net model. However, the performance of the model diminishes in the presence of artifacts, such as image reflection …

Calcium Analysis Of Intercellular Communications Between Alpha Cells And Delta Cells Across The Pancreatic Islet, Shichao Gao

McKelvey School of Engineering Graduate Student Theses & Dissertations

The intricate communication between different cell types within pancreatic islets plays a crucial role in regulating glucose homeostasis. Among these cellular interactions, the interplay between α cells (responsible for glucagon secretion) and δ cells (secreting somatostatin) is particularly intriguing due to the inhibitory effects of somatostatin on glucagon release. Understanding the intercellular interactions between α and δ cells may provide insights into the mechanisms underlying the coordinated regulation of blood glucose levels. In this study, we employed a multi-modal approach combining confocal imaging, image analysis, and correlation network analysis to investigate the spatial relationships and potential functional connections between α …

Structured Light Radial-View Endoscope Imaging Development For Evaluation Of Human Tissue, Xiyan Li

McKelvey School of Engineering Graduate Student Theses & Dissertations

In this thesis, we improved an existing structured light methodology to more accurately estimate tissue optical properties imaged in a single snapshot. The developed approach is a major enhancement of the traditional three-phase spatial frequency domain imaging system: It greatly simplifies the operational procedure while also maintaining high accuracy in optical property measurements .

Leveraging this improved approach, we have developed a low-cost, compact, and radial-view endoscope prototype. This prototype combines affordability with high functionality and a user-friendly design. To assess the performance and reliability of our endoscope, we conducted comprehensive evaluations involving tissue-mimicking phantoms, as well as ex vivo …

Evaluating Neuroimaging Modalities In The A/T/N Framework: Single And Combined Fdg-Pet And T1-Weighted Mri For Alzheimer’S Diagnosis, Peiwang Liu

McKelvey School of Engineering Graduate Student Theses & Dissertations

With the escalating prevalence of dementia, particularly Alzheimer's Disease (AD), the need for early and precise diagnostic techniques is rising. This study delves into the comparative efficacy of Fluorodeoxyglucose Positron Emission Tomography (FDG-PET) and T1-weighted Magnetic Resonance Imaging (MRI) in diagnosing AD, where the integration of multimodal models is becoming a trend. Leveraging data from the Alzheimer's Disease Neuroimaging Initiative (ADNI), we employed linear Support Vector Machines (SVM) to assess the diagnostic potential of these modalities, both individually and in combination, within the AD continuum. Our analysis, under the A/T/N framework's 'N' category, reveals that FDG-PET consistently outperforms T1w-MRI across …

Resolving The Nanoscale Structure Of Β-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation–Localization Microscopy, Weiyan Zhou, Conor L. O'Neill, Tianben Ding, Oumeng Zhang, Jai S. Rudra, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Synthetic peptides that self-assemble into cross-β fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8^L and KFE8^D and the pathological amyloid-beta peptide Aβ42. Importantly, NR SMOLM reveals the helical (bilayer) …

6d Single-Fluorogen Orientation-Localization Microscopy For Elucidating The Architecture Of Beta-Sheet Assemblies And Biomolecular Condensates, Tingting Wu, Weiyan Zhou, Jai S. Rudra, Rohit V. Pappu, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

We develop six-dimensional single-molecule orientation-localization microscopy (SMOLM) to measure the 3D positions and 3D orientations simultaneously of single fluorophores. We show how careful optimization of phase and polarization modulation components can encode phase, polarization, and angular spectrum information from each fluorescence photon into a microscope’s dipole-spread function. We used the transient binding and blinking of Nile red (NR) to characterize the helical structure of fibrils formed by designed amphipathic peptides, KFE8^L and KFE8^D, and the pathological amyloid-beta peptide Aβ42. We also deployed merocyanine 540 to uncover the interfacial architectures of biomolecular condensates.

Single-Molecule Super-Resolution Imaging Of Geobacter Sulfurreducens Under Anaerobic Conditions, Ziyi Hu

McKelvey School of Engineering Graduate Student Theses & Dissertations

Geobacter sulfurreducens are anaerobic bacteria capable of making electrical contacts with other organisms and extracellular electron acceptors. The challenge of imaging live Geobacter bacteria is maintaining anaerobic conditions during the imaging process. In this thesis, we augment a single-molecule localization microscope (SMLM) with a home-built anaerobic imaging chamber and use constant argon bubbling to maintain oxygen-free imaging conditions. To validate the imaging protocol, we use the transient binding of Nile red to resolve the spherical morphology of lipid-coated glass spheres with nanoscale resolution. However, when imaging Geobacter, the distribution of Nile red localizations is non-uniform, both between different cells …

Development Of A Multispectral Vis-Swir Imaging Modality For Cutaneous Water Assessment, Quinlan Mcgrath

McKelvey School of Engineering Graduate Student Theses & Dissertations

Inflammatory skin diseases are estimated to impact 20% of the global population and are the fourth leading cause of nonfatal disability worldwide. Diagnosis and management are predominantly based on clinician visual assessment of disease related changes in skin morphology. The qualitative nature of this method can result in misdiagnosis and underdiagnoses of treatable diseases. There persists systematic undertreatment of skin of color patients given the more subtle presentation of erythema against pigmented skin. There exists a clinical need for a quantitative and objective inflammation assessment tool that meets the needs of a diverse patient population. The accumulation of interstitial fluid …

Dual Color Optogenetic Control For Analyzing Cardiac Function In Drosophila, Jiantao Zhu

McKelvey School of Engineering Graduate Student Theses & Dissertations

Prolonged consumption of carbohydrate-rich diets and immobile lifestyles frequently cause metabolic disorders and obesity and, as a result, may lead to progressive heart dysfunction among broad social groups of the population. Drosophila melanogaster serves as an essential model organism in cardiovascular disease research due to conserved physiological and genomic traits shared with humans, its genetic and molecular toolbox versatility, and cost-effective maintenance. Here, we combine optogenetics and optical coherence tomography to study cardiovascular function in D. melanogaster. A new optogenetic pacing system has been developed, employing a transgenic line carrying two opsins: ChR2 and NpHR2.0. A custom-built hardware setup …

Synthesis, Radiolabeling And Evaluation Of A Suite Of Tracers With 44Sc For Detecting Extracellular Dna, Zhiyao Li

McKelvey School of Engineering Graduate Student Theses & Dissertations

Neutrophil extracellular traps involve the rapid translocation of DNA to the outside of the cell under certain stimuli. This structure forms a fibrous network that is able to limit the spread of pathogens and to kill microorganisms. It has also been shown to be present in various pathological processes such as inflammation, autoimmune diseases, and cancer metastasis. Currently, the formation process of NETs in vivo is being extensively studied. However noninvasive detection and quantitation has yet to be achieved. A class of PET tracers are described here that consists of a DNA dye as the backbone that is labeled with …

Six-Dimensional Single-Molecule Imaging With Isotropic Resolution Using A Multi-View Reflector Microscope, Oumeng Zhang, Zijian Guo, Yuanyuan He, Tingting Wu, Michael D. Vahey, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Imaging of both the positions and orientations of single fluorophores, termed single-molecule orientation-localization microscopy, is a powerful tool for the study of biochemical processes. However, the limited photon budget associated with single-molecule fluorescence makes high-dimensional imaging with isotropic, nanoscale spatial resolution a formidable challenge. Here we realize a radially and azimuthally polarized multi-view reflector (raMVR) microscope for the imaging of the three-dimensional (3D) positions and 3D orientations of single molecules, with precisions of 10.9 nm and 2.0° over a 1.5-μm depth range. The raMVR microscope achieves 6D super-resolution imaging of Nile red molecules transiently bound to lipid-coated spheres, accurately resolving …

Development Of Optical Coherence Tomography Angiography System For Mouse Eye Imaging And Measurement Of Vasodilation, Yilin Li

McKelvey School of Engineering Graduate Student Theses & Dissertations

Optical coherence tomography angiography (OCTA) is broadly known as a non-invasive technology that allows examining the retinal and choroidal vasculatures, alternative to fluorescein angiography. In this study, we have developed an optical coherence tomography (OCT) system for imaging the mouse eye. We measure the retinal thickness, which is 216.53um. We apply OCTA to characterize the quantitative change in vessel diameter and the perfusion density of the retina after the wild-type mice are administered the adenosine or inhale carbon dioxide. Comparing the images acquired one minute and 5 minutes after the injection or CO₂ inhalation with the baseline, distinct changes …

Machine Learning And Scalable Informatics Methods To Predict Disease Status From Multimodal Biomedical Data, Hossein Mohammadian Foroushani

McKelvey School of Engineering Graduate Student Theses & Dissertations

Biological understanding of complex diseases such as stroke and obesity is critical for the advancement of medicine. Further knowledge discovery can provide effective biomarkers to improve disease diagnosis and prognosis, identify driver mutations, predict individual genetic susceptibility for early prevention and effective disease management, and facilitate development of personalized drugs. Stroke is the second leading cause of death and long-term disability in the world. Thus, stroke management is a time-sensitive emergency. The initial hours after stroke onset map the trajectory of subsequent neurologic complications. Cerebral edema develops hours to days after acute ischemic stroke and may result in midline shift …

Evaluation And Clinical Implementation Of A Dual-Energy Ct Stopping-Power Ratio Mapping Technique For Proton-Therapy Treatment Planning, Maria Jose Medrano Matamoros

McKelvey School of Engineering Graduate Student Theses & Dissertations

Proton radiotherapy has the potential to treat tumors with better conformal dose distribution than competing modalities when the rapid dose falloff at the end of the proton-beam range is correctly aligned to the edge of the clinical target volume (CTV). However, its clinical potential is dependent on the accurate localization of the Bragg-peak position from predicted stopping-power ratio maps. The method that is most commonly used in today’s clinical practice for predicting stopping-power ratio (SPR) consists of a stoichiometric calibrationtechnique based on single-energy CT (SECT) for direct estimation of patient-specific SPR distribution from vendor-reconstructed Hounsfield Unit (HU) images. Unfortunately, this …

Advancing The Portability, Field Of View, And Spatiotemporal Resolution Of Multi-Parametric Photoacoustic Microscopy, Fenghe Zhong

McKelvey School of Engineering Graduate Student Theses & Dissertations

Capable of structural, functional, molecular, and metabolic imaging with high spatial resolution in vivo, photoacoustic microscopy (PAM) is an emerging tool in biomedical research. Further, recent advances in multi-parametric acquisition and analysis make PAM uniquely capable of simultaneously mapping the total concentration of hemoglobin (CHb), oxygen saturation of hemoglobin (sO2), and blood flow speed. However, there are multiple major limitations, including, the large footprint, low imaging speed, limited imaging contrast, and insufficient axial resolution, which impedance its applications in basic and translational research. In this dissertation, I presented potential solutions to overcome the limitations above.In the first part of this …

Resolving The Three-Dimensional Rotational And Translational Dynamics Of Single Molecules Using Radially And Azimuthally Polarized Fluorescence, Oumeng Zhang, Weiyan Zhou, Jin Lu, Tingting Wu, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

We report a radially and azimuthally polarized (raPol) microscope for high detection and estimation performance in single-molecule orientation-localization microscopy (SMOLM). With 5000 photons detected from Nile red (NR) transiently bound within supported lipid bilayers (SLBs), raPol SMOLM achieves 2.9 nm localization precision, 1.5° orientation precision, and 0.17 sr precision in estimating rotational wobble. Within DPPC SLBs, SMOLM imaging reveals the existence of randomly oriented binding pockets that prevent NR from freely exploring all orientations. Treating the SLBs with cholesterol-loaded methyl-β-cyclodextrin (MβCD-chol) causes NR’s orientational diffusion to be dramatically reduced, but curiously NR’s median lateral displacements drastically increase from 20.8 to …

Single-Molecule Localization Microscopy Of 3d Orientation And Anisotropic Wobble Using A Polarized Vortex Point Spread Function, Tianben Ding, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule’s wobble, simultaneously from a single image. Even when imaging dim emitters (∼500 photons detected), the polarized vortex PSF can obtain 12 nm localization precision, 4°–8° orientation precision, and 26° wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics …

Using Computer Vision To Track Anatomical Structures During Cochlear Implant Surgery, Nicholas Bach

McKelvey School of Engineering Graduate Student Theses & Dissertations

There is a steep learning curve for surgeons performing cochlear implant surgeries. We aimed to use computer vision to track anatomical features with the goal of helping surgeons perform cochlear implant surgery without damaging the cochlea. We compared nine algorithms in total, seven object tracking algorithms and two optical flow algorithms utilizing the LucasKanade method, on manually created cochlear implant surgery videos to determine the accuracy associated with each. Compared with eight other algorithms, we observed that an iterative pyramidal implementation of the Lucas-Kanade (IPLK) method, implemented through OpenCV, performed the best. The IPLK method had the lowest error rate …

Injectable Ct/Mri Contrast Agent For Gastrointestinal Tumor Tracking, Luna Zhang

McKelvey School of Engineering Graduate Student Theses & Dissertations

Gastrointestinal cancers remain to be of the most common and deadly cancers worldwide. Early detection and treatments are crucial for reducing mortality and improving patient outcome. Radiation therapy is a non-invasive localized tumor treatment method, and utilizes radiation to kill the cancerous cells and shrink tumors at specific sites. Precise localization at the target tumor site is therefore important before radiation therapy, especially for gastrointestinal tumor sites located in the moving bowel. Currently, invasive endoscopies along with ink tattoos are used for identifying tumor location, which often require sedation and bring much discomfort. Imaging tests, including CT and MRI, play …

Computational Modelling Enables Robust Multidimensional Nanoscopy, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

The following sections are included:

• Present State of Computational Modelling in Fluorescence Nanoscopy

• Recent Contributions to Computational Modelling in Fluorescence Nanoscopy

• Outlook on Computational Modelling in Fluorescence Nanoscopy

• Acknowledgments

• References

Focused Ultrasound-Mediated Drug Delivery To The Brainstem, Dezhuang Ye

McKelvey School of Engineering Graduate Student Theses & Dissertations

Brainstem gliomas are tumors that occur in the brainstem, the brain region that connects the brain to the spinal cord and controls vital body functions. The critical anatomic location of the brainstem precludes surgical intervention and limits the use of invasive therapeutic techniques. Moreover, the frequently intact blood-brain barrier (BBB) of most brainstem gliomas prevents therapeutic agents from reaching the diseased site. The currently available techniques for brain drug delivery are either invasive (e.g., convection-enhanced delivery) or lack targeting to the diseased site (e.g., intranasal brain drug delivery). Novel techniques that can noninvasively overcome the BBB are critically needed for …