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.

Computational Design Of Fiber-Optic Probes For Biosensing, Suwarna Karna

Electrical Engineering Theses

This thesis presents a study on the optical characteristics of hollow-core photonic crystal fibers (HC-PCFs) with a band gap cladding structure and their applications in optical fiber sensing. This 800B HC-PCF exhibited excellent optical properties and has a flexible structure, which makes them suitable for a wide range of industrial applications. Finite element simulations and structural optimization designs were conducted using the surface plasmon resonance (SPR) technique to determine the optimal performance parameters of the 800B HC-PCF. The fiber was further modified using the SPR technique to improve its practical detection capabilities. The performance of the modified fiber was observed …

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 …

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 …

Development Of Quantitative Ultrasound-Mediated Molecular Imaging Of The Tumor Microenvironment, Trevor Mitcham

Dissertations & Theses (Open Access)

While conventional diagnostic imaging modalities provide anatomical information to clinicians, these techniques are not sensitive to critical physiological processes. In order to properly classify cancer, it is necessary to investigate noninvasive methods which can provide insight into these processes, allowing clinicians to determine personalized therapeutic options. Therefore, molecular imaging is focused on visualization and characterization of biomarkers within the tumor microenvironment (TME), which can then be combined with the anatomical information provided from diagnostic imaging.

Two such biomarkers of interest are blood oxygen saturation (SO₂) and cell receptor expression. SO₂ is a measure of the fraction of …

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

Clinical Applications And Feasibility Of Proton Ct And Proton Radiography, Christina Marie Sarosiek

Graduate Research Theses & Dissertations

Proton therapy is a form of radiation treatment for cancer that utilizes the Bragg peak to create conformal high dose regions around the tumor volume. However, the use of x-ray computed tomography (CT) and x-ray radiography for treatment planning and pre-treatment quality assurance procedures improves the achievable effectiveness of proton treatment plans (using proton CT) and the pretreatment verification (using proton radiography). Errors in the conversion from x-ray Hounsfield units (HU) to proton relative stopping powers (RSP) leads to errors in the predicted proton range. To account for the errors, 3.5% margins are included in the treatment plan. This means …

Combining Cryo-Em Density Map And Residue Contact For Protein Secondary Structure Topologies, Maytha Alshammari, Jing He

Computer Science Faculty Publications

Although atomic structures have been determined directly from cryo-EM density maps with high resolutions, current structure determination methods for medium resolution (5 to 10 Å) cryo-EM maps are limited by the availability of structure templates. Secondary structure traces are lines detected from a cryo-EM density map for α-helices and β-strands of a protein. A topology of secondary structures defines the mapping between a set of sequence segments and a set of traces of secondary structures in three-dimensional space. In order to enhance accuracy in ranking secondary structure topologies, we explored a method that combines three sources of information: a set …

Approaches To Studying Bacterial Biofilms In The Bioeconomy With Nanofabrication Techniques And Engineered Platforms., Michelle Caroline Halsted

Doctoral Dissertations

Studies that estimate more than 90% of bacteria subsist in a biofilm state to survive environmental stressors. These biofilms persist on man-made and natural surfaces, and examples of the rich biofilm diversity extends from the roots of bioenergy crops to electroactive biofilms in bioelectrochemical reactors. Efforts to optimize microbial systems in the bioeconomy will benefit from an improved fundamental understanding of bacterial biofilms. An understanding of these microbial systems shows promise to increase crop yields with precision agriculture (e.g. biosynthetic fertilizer, microbial pesticides, and soil remediation) and increase commodity production yields in bioreactors. Yet conventional laboratory methods investigate these micron-scale …

3-D Fabry–Pérot Cavities Sculpted On Fiber Tips Using A Multiphoton Polymerization Process, Jonathan W. Smith, Jeremiah C. Williams, Joseph S. Suelzer, Nicholas G. Usechak, Hengky Chandrahalim

Faculty Publications

This paper presents 3-D Fabry–Pérot (FP) cavities fabricated directly onto cleaved ends of low-loss optical fibers by a two-photon polymerization (2PP) process. This fabrication technique is quick, simple, and inexpensive compared to planar microfabrication processes, which enables rapid prototyping and the ability to adapt to new requirements. These devices also utilize true 3-D design freedom, facilitating the realization of microscale optical elements with challenging geometries. Three different device types were fabricated and evaluated: an unreleased single-cavity device, a released dual-cavity device, and a released hemispherical mirror dual-cavity device. Each iteration improved the quality of the FP cavity's reflection spectrum. The …

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 …

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 …

Incorporating Cardiac Substructures Into Radiation Therapy For Improved Cardiac Sparing, Eric Daniel Morris

Wayne State University Dissertations

Growing evidence suggests that radiation therapy (RT) doses to the heart and cardiac substructures (CS) are strongly linked to cardiac toxicities, though only the heart is considered clinically. This work aimed to utilize the superior soft-tissue contrast of magnetic resonance (MR) to segment CS, quantify uncertainties in their position, assess their effect on treatment planning and an MR-guided environment.

Automatic substructure segmentation of 12 CS was completed using a novel hybrid MR/computed tomography (CT) atlas method and was improved upon using a 3-dimensional neural network (U-Net) from deep learning. Intra-fraction motion due to respiration was then quantified. The inter-fraction setup …

Gravity-Drawing Flexible Silicone Filaments As Fiber Optics And Model Foldamers, Katherine Snell

CMC Senior Theses

Here, we present a method of gravity-drawing polydimethylsiloxane (PDMS) silicone fibers with application as fiber optics and as model foldamers. Beginning as a viscous liquid, PDMS is cured using heat until its measured viscosity reaches 4000 mPa•s. The semi-cured elastomer is then extruded through a tube furnace to produce thin (diameters on the order of hundred micrometers) filaments with scalable lengths. PDMS is biocompatible, gas-permeable, flexible, and hydrophobic. Additionally, the PDMS surface hydrophobicity can be modified via UV exposure, O2 plasma, and corona discharge. We demonstrate the patternibility (i.e patterns of hydrophobicity) of PDMS fibers, adding complexity to potential foldamer …

Seeing Eye To Eye: A Machine Learning Approach To Automated Saccade Analysis, Maigh Attre

Honors Scholar Theses

Abnormal ocular motility is a common manifestation of many underlying pathologies particularly those that are neurological. Dynamics of saccades, when the eye rapidly changes its point of fixation, have been characterized for many neurological disorders including concussions, traumatic brain injuries (TBI), and Parkinson’s disease. However, widespread saccade analysis for diagnostic and research purposes requires the recognition of certain eye movement parameters. Key information such as velocity and duration must be determined from data based on a wide set of patients’ characteristics that may range in eye shapes and iris, hair and skin pigmentation [36]. Previous work on saccade analysis has …

Applications Of Machine Learning In Nuclear Imaging And Radiation Detection, Shaikat Mahmood Galib

Doctoral Dissertations

"The main focus of this work is to use machine learning and data mining techniques to address some challenging problems that arise from nuclear data. Specifically, two problem areas are discussed: nuclear imaging and radiation detection. The techniques to approach these problems are primarily based on a variant of Artificial Neural Network (ANN) called Convolutional Neural Network (CNN), which is one of the most popular forms of 'deep learning' technique.

The first problem is about interpreting and analyzing 3D medical radiation images automatically. A method is developed to identify and quantify deformable image registration (DIR) errors from lung CT scans …

Optical Vortex And Poincaré Analysis For Biophysical Dynamics, Anindya Majumdar

Dissertations, Master's Theses and Master's Reports

Coherent light - such as that from a laser - on interaction with biological tissues, undergoes scattering. This scattered light undergoes interference and the resultant field has randomly added phases and amplitudes. This random interference pattern is known as speckles, and has been the subject of multiple applications, including imaging techniques. These speckle fields inherently contain optical vortices, or phase singularities. These are locations where the intensity (or amplitude) of the interference pattern is zero, and the phase is undefined.

In the research presented in this dissertation, dynamic speckle patterns were obtained through computer simulations as well as laboratory setups …

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 …

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 …

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 …

Characterization Of Computed Tomography Radiomic Features Using Texture Phantoms, Muhammad Shafiq Ul Hassan

USF Tampa Graduate Theses and Dissertations

Radiomics treats images as quantitative data and promises to improve cancer prediction in radiology and therapy response assessment in radiation oncology. However, there are a number of fundamental problems that need to be solved in order to potentially apply radiomic features in clinic. The first basic step in computed tomography (CT) radiomic analysis is the acquisition of images using selectable image acquisition and reconstruction parameters. Radiomic features have shown large variability due to variation of these parameters. Therefore, it is important to develop methods to address these variability issues in radiomic features due to each CT parameter. To this end, …

System Optimization And Iterative Image Reconstruction In Photoacoustic Computed Tomography For Breast Imaging, Yang Lou

McKelvey School of Engineering Theses & Dissertations

Photoacoustic computed tomography(PACT), also known as optoacoustic tomography (OAT), is an emerging imaging technique that has developed rapidly in recent years. The combination of the high optical contrast and the high acoustic resolution of this hybrid imaging technique makes it a promising candidate for human breast imaging, where conventional imaging techniques including X-ray mammography, B-mode ultrasound, and MRI suffer from low contrast, low specificity for certain breast types, and additional risks related to ionizing radiation. Though significant works have been done to push the frontier of PACT breast imaging, it is still challenging to successfully build a PACT breast imaging …

Quantitative Optical Studies Of Oxidative Stress In Rodent Models Of Eye And Lung Injuries, Zahra Ghanian

Theses and Dissertations

Optical imaging techniques have emerged as essential tools for reliable assessment of organ structure, biochemistry, and metabolic function. The recognition of metabolic markers for disease diagnosis has rekindled significant interest in the development of optical methods to measure the metabolism of the organ.

The objective of my research was to employ optical imaging tools and to implement signal and image processing techniques capable of quantifying cellular metabolism for the diagnosis of diseases in human organs such as eyes and lungs. To accomplish this goal, three different tools, cryoimager, fluorescent microscope, and optical coherence tomography system were utilized to study the …

Increasing 18f-Fdg Pet/Ct Capabilities In Radiotherapy For Lung And Esophageal Cancer Via Image Feature Analysis, Jasmine Alexandria Oliver

USF Tampa Graduate Theses and Dissertations

Positron Emission Tomography (PET) is an imaging modality that has become increasingly beneficial in Radiotherapy by improving treatment planning (1). PET reveals tumor volumes that are not well visualized on computed tomography CT or MRI, recognizes metastatic disease, and assesses radiotherapy treatment (1). It also reveals areas of the tumor that are more radiosensitive allowing for dose painting - a non-homogenous dose treatment across the tumor (1). However, PET is not without limitations. The quantitative unit of PET images, the Standardized Uptake Value (SUV), is affected by many factors such as reconstruction algorithm, patient weight, and tracer uptake time (2). …

Characterization Of Low Density Intracranial Lesions Using Dual-Energy Computed Tomography, Jessica L. Nute

Dissertations & Theses (Open Access)

Calcific and hemorrhagic foci of susceptibility are frequently encountered on routine brain MR studies. Both etiologies cause variations in local magnetic field strength, leading to dark regions on the MR images that cannot be classified. Single-energy CT (SECT) can be used to identify lesions with attenuation over 100 HU as calcific, however lesions with lower attenuation cannot be reliably identified. While calcific lesions are unlikely to cause harm, hemorrhagic lesions carry a risk of subsequent intracranial bleeding; as such, identification of hemorrhage is vital in preventing the inappropriate use of anticoagulant medications in patients with hemorrhagic lesions.

Given there currently …

Frequency Domain Ultrasound Waveform Tomography Breast Imaging, Gursharan Singh Sandhu

Wayne State University Dissertations

Ultrasound tomography is an emerging modality for imaging breast tissue for the detection of disease. Using the principles of full waveform inversion, high-resolution quantitative sound speed and attenuation maps of the breast can be created. In this thesis, we introduce some basic principles of imaging breast disease and the formalism of sound wave propagation. We present numerical methods to model acoustic wave propagation as well methods to solve the corresponding inverse problem. Numerical simulations of sound speed and attenuation reconstructions are used to assess the efficacy of the algorithm. A careful review of the preprocessing techniques needed for the successful …