Bioimaging and Biomedical Optics Commons^™

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.

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

McKelvey School of Engineering 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 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 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 …

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 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 …

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 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 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 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 …

Single‐Molecule 3d Orientation Imaging Reveals Nanoscale Compositional Heterogeneity In Lipid Membranes, Jin Lu, Hesam Mazidi, Tianben Ding, Oumeng Zhang, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules μm⁻²) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single‐molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve …

Pathophysiology And Proteogenomics Of Post-Infectious And Post-Hemorrhagic Hydrocephalus In Infants, Albert M. Isaacs

Arts & Sciences Electronic Theses and Dissertations

Post-infectious (PIH) and post-hemorrhagic (PHH) hydrocephalus occur as sequalae of neonatal sepsis or intraventricular hemorrhage (IVH) of prematurity, respectively. Together, PIH and PHH represent the most common form of infantile hydrocephalus, the most common indication for neurosurgery in children globally, and the leading cause of neurological morbidity and mortality worldwide. The lack of understanding of the pathophysiology of PIH and PHH, particularly with regards to the host central nervous system response to the antecedent infection and hemorrhage, perturbation of differentiating neural stems in the ventricular (VZ) and subventricular (SVZ) zones, and damage to periventricular white matter (PVWM) tracts carrying sensorimotor …

Phantoms To Placentas: Mr Methods For Oxygen Quantification, Kelsey Meinerz

Arts & Sciences Electronic Theses and Dissertations

Molecular oxygen (O2) is vital for efficient energy production and improper oxygenation is a hallmark of disease or metabolic dysfunction. In many pathologies, knowledge of tissue oxygen levels (pO2) could aid in diagnosis and treatment planning. The gold standard for pO2 measures in tissue are implantable probes, which are invasive, require surgery for placement, and are inaccessible to certain regions of the body. Methods for determining pO2 both non-invasively and quantitatively are lacking. The slight paramagnetic nature of O2 provides opportunities to non-invasively characterize pO2 in tissue via magnetic resonance (MR) techniques. As such, O2 can be treated as a …

Computational Imaging Methods For Analysis Of Datscan Spect Images, Hae Sol Moon

McKelvey School of Engineering Theses & Dissertations

There is an important need to develop biomarkers to improve the diagnosis and assess the severity of Parkinson’s disease (PD). The potential to derive such biomarkers from quantitative dopamine transporter scan (DaT-Scan) single-photon emission computed tomography (SPECT) imaging, in particular the uptake of DaT in the caudate, putamen, and globus pallidus regions, is highly appealing as imaging is non-invasive and DaTScan is already used in the management of patients with PD. However, reliable quantification requires reliable segmentation of these regions in these images. Reliable segmentation is challenging due to the limited spatial resolution and high image noise in SPECT images …

Multi-Dimensional Extension Of The Alternating Minimization Algorithm In X-Ray Computed Tomography, Jingwei Lu

McKelvey School of Engineering Theses & Dissertations

X-ray computed tomography (CT) is an important and effective tool in medical and industrial imaging applications. The state-of-the-art methods to reconstruct CT images have had great development but also face challenges. This dissertation derives novel algorithms to reduce bias and metal artifacts in a wide variety of imaging modalities and increase performance in low-dose scenarios. The most widely available CT systems still use the single-energy CT (SECT), which is good at showing the anatomic structure of the patient body. However, in SECT image reconstruction, energy-related information is lost. In applications like radiation treatment planning and dose prediction, accurate energy-related information …

Multi-Dimensional Extension Of The Alternating Minimization Algorithm In X-Ray Computed Tomography, Jingwei Lu

McKelvey School of Engineering Theses & Dissertations

X-ray computed tomography (CT) is an important and effective tool in medical and industrial

imaging applications. The state-of-the-art methods to reconstruct CT images have had

great development but also face challenges. This dissertation derives novel algorithms to

reduce bias and metal artifacts in a wide variety of imaging modalities and increase performance

in low-dose scenarios.

The most widely available CT systems still use the single-energy CT (SECT), which is

good at showing the anatomic structure of the patient body. However, in SECT image

reconstruction, energy-related information is lost. In applications like radiation treatment

planning and dose prediction, accurate energy-related information …

Quantitatively Studying Tissue Damage In Multiple Sclerosis Using Gradient Recalled Echo Mri Sequences, Biao Xiang

Arts & Sciences Electronic Theses and Dissertations

Multiple Sclerosis (MS) is an unpredictable, often disabling disease of the central nervous system (CNS) that disrupts the flow of information within the brain, and between the brain the body. MS is the most common progressive neurologic disease of young adults, affecting approximately 2.3 million people worldwide. It is estimated that more than 700,000 individuals are affected by MS in United States. While MS has been studied for decades, the cause of it is still not definite and a fully effective treatment for MS is not yet available.

Magnetic resonance imaging (MRI) has been used extensively in MS diagnosis and …

Quantitatively Studying Tissue Damage In Multiple Sclerosis Using Gradient Recalled Echo Mri Sequences, Biao Xiang

Arts & Sciences Electronic Theses and Dissertations

Multiple Sclerosis (MS) is an unpredictable, often disabling disease of the central nervous system (CNS) that disrupts the flow of information within the brain, and between the brain the body. MS is the most common progressive neurologic disease of young adults, affecting approximately 2.3 million people worldwide. It is estimated that more than 700,000 individuals are affected by MS in United States. While MS has been studied for decades, the cause of it is still not definite and a fully effective treatment for MS is not yet available. Magnetic resonance imaging (MRI) has been used extensively in MS diagnosis and …

Preclinical Imaging Of Multiple Myeloma Therapy Response, Deep Hathi

McKelvey School of Engineering Theses & Dissertations

Multiple myeloma (MM) is a debilitating hematologic malignancy of terminally differentiated plasma cells in the bone marrow (BM). Advances in therapeutic regimens and the use of autologous stem cell transplantation have significantly improved survival rates and quality of life in patients. However, the disease remains incurable, with shorter, successive remission cycles following relapse. To reduce systemic, off-target toxicity and improve quality of life, there is a need for improved stratification of responding patients. Identification of specific, noninvasive, imaging biomarkers that correlate to therapeutic efficacy is an attractive strategy for stratifying responding patients, since the use of positron emission tomography (PET), …

Regularized Fourier Ptychographic Microscopy, Shiqi Xu

McKelvey School of Engineering Theses & Dissertations

Quantitative phase image (QPI) is a popular microscopy technique for studying cell morphology. Recently, Fourier ptychographic microscopy (FPM) has emerged as a low-cost computational microscopy technique for forming high-resolution wide-field QPI images by taking multiple images from different illumination angles. However, the applicability of FPM to dynamic imaging is limited by its high data requirement. In this thesis, we propose new methods for highly compressive FPM imaging using a data-adaptive sparse coding and an online plug-and-play (PnP) method with non-local priors based on the fast iterative shrinkage/threshold algorithm (FISTA). We validate the proposed method on both simulated and experimental data …

Long-Term, Super-Resolution Imaging Of Amyloid Structures Using Transient Amyloid Binding Microscopy, Tianben Ding, Kevin Spehar, Jan Bieschke, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Amyloid fibrils and tangles are signatures of Alzheimer disease, but nanometer-sized aggregation intermediates are hypothesized to be the structures most toxic to neurons. The structures of these oligomers are too small to be resolved by conventional light microscopy. We have developed a simple and versatile method, called transient amyloid binding (TAB), to image amyloid structures with nanoscale resolution using amyloidophilic dyes, such as Thioflavin T, without the need for covalent labeling or immunostaining of the amyloid protein. Transient binding of ThT molecules to amyloid structures over time generates photon bursts that are used to localize single fluorophores with nanometer precision. …

Basis Vector Model Method For Proton Stopping Power Estimation Using Dual-Energy Computed Tomography, Shuangyue Zhang

McKelvey School of Engineering Theses & Dissertations

Accurate estimation of the proton stopping power ratio (SPR) is important for treatment planning and dose prediction for proton beam therapy. The state-of-the-art clinical practice for estimating patient-specific SPR distributions is the stoichiometric calibration method using single-energy computed tomography (SECT) images, which in principle may introduce large intrinsic uncertainties into estimation results. One major factor that limits the performance of SECT-based methods is the Hounsfield unit (HU) degeneracy in the presence of tissue composition variations. Dual-energy computed tomography (DECT) has shown the potential of reducing uncertainties in proton SPR prediction via scanning the patient with two different source energy spectra. …

Radiolabeled Nanohydroxyapatite As A Platform For The Development Of New Pet Imaging Agents, Stacy Lee Queern

Arts & Sciences Electronic Theses and Dissertations

Positron emission tomography (PET) imaging utilizes drugs labeled with positron emitters to target and evaluate different biological processes occurring in the body. Tailoring medicine to the individual allows for higher quality of care with better diagnosis and treatment and is a key purpose for advancing research into developing new platforms for PET imaging agents. A PET nuclide of high interest for the development of these agents is 89Zr. This can be attributed to the long half-life of 3.27 days and low positron energy of 89Zr.

In this work, we developed a production method for 89Zr using Y sputtered coins that …

In Vivo Vascular Imaging With Photoacoustic Microscopy, Hsun-Chia Hsu

McKelvey School of Engineering Theses & Dissertations

Photoacoustic (PA) tomography (PAT) has received extensive attention in the last decade for its capability to provide label-free structural and functional imaging in biological tissue with highly scalable spatial resolution and penetration depth. Compared to modern optical modalities, PAT offers speckle-free images and is more sensitive to optical absorption contrast (with 100% relative sensitivity). By implementing different regimes of optical wavelength, PAT can be used to image diverse light-absorbing biomolecules. For example, hemoglobin is of particular interest in the visible wavelength regime owing to its dominant absorption, and lipids and water are more commonly studied in the near-infrared regime.

In …

Super‐Resolution Imaging Of Amyloid Structures Over Extended Times By Using Transient Binding Of Single Thioflavin T Molecules, Kevin Spehar, Tianben Ding, Yuanzi Sun, Niraja Kedia, Jin Lu, George R. Nahass, Matthew D. Lew, Jan Bieschke

Electrical & Systems Engineering Publications and Presentations

Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super‐resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics …

Developing Wavefront Shaping Techniques For Focusing Through Highly Dynamic Scattering Media, Ashton Hemphill

McKelvey School of Engineering Theses & Dissertations

One of the prime limiting factors of optical imaging in biological applications is the diffusion of light by tissue, which prevents focusing at depths greater than the optical diffusion limit of ~1 mm in soft tissue. This greatly restricts the utility of optical diagnostic and therapeutic techniques, such as optogenetics, microsurgery, optical tweezing, and phototherapy of deep tissue, which require focused light in order to function. Wavefront shaping extends the depth at which optical focusing may be achieved by compensating for phase distortions induced by scattering, allowing for focusing through constructive interference.

However, due to physiological motion, scattering of light …

Fluorescence Guided Tumor Imaging: Foundations For Translational Applications, Jessica P. Miller

McKelvey School of Engineering Theses & Dissertations

Optical imaging for medical applications is a growing field, and it has the potential to improve medical outcomes through its increased sensitivity and specificity, lower cost, and small instrumentation footprint as compared to other imaging modalities. The method holds great promise, ranging from direct clinical use as a diagnostic or therapeutic tool, to pre-clinical applications for increased understanding of pathology. Additionally, optical imaging uses non-ionizing radiation which is safe for patients, so it can be used for repeated imaging procedures to monitor therapy, guide treatment, and provide real-time feedback. The versatile features of fluorescence-based optical imaging make it suited for …

Developing Photoacoustic Tomography Devices For Translational Medicine And Basic Science Research, Tsz Wai Wong

McKelvey School of Engineering Theses & Dissertations

Photoacoustic (PA) tomography (PAT) provides volumetric images of biological tissue with scalable spatial resolutions and imaging depths, while preserving the same imaging contrast—optical absorption. Taking the advantage of its 100% sensitivity to optical absorption, PAT has been widely applied in structural, functional, and molecular imaging, with both endogenous and exogenous contrasts, at superior depths than pure optical methods. Intuitively, hemoglobin has been the most commonly studied biomolecule in PAT due to its strong absorption in the visible wavelength regime.

One of the main focuses of this dissertation is to investigate an underexplored wavelength regime—ultraviolet (UV), which allows us to image …