Biomedical Engineering and Bioengineering 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.

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 …

Plasmonic Nanomaterials-Based Point-Of-Care Biosensors, Rohit Gupta

McKelvey School of Engineering Theses & Dissertations

Point-of-care (POC) biosensors, although rapid and easy-to-use, are orders magnitude less sensitive than laboratory-based tests. Further they are plagued by poor stability of recognition element thus limiting its widespread applicability in resource-limited settings. Therefore, there is a critical need for realizing stable POC biosensors with sensitivity comparable to gold-standard laboratory-based tests. This challenge constitutes the fundamental basis of this dissertation work– to expand access to quality and accurate biodiagnostic tools. At the heart of these solutions lies plasmonic nanoparticles which exhibit unique optical properties which are attractive for label-free and labelled biosensors.Firstly, we improve the stability and applicability of label-free …

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 …

Assessment And Diagnosis Of Human Colorectal And Ovarian Cancer Using Optical Imaging And Computer-Aided Diagnosis, Yifeng Zeng

McKelvey School of Engineering Theses & Dissertations

Tissue optical scattering has recently emerged as an important diagnosis parameter associated with early tumor development and progression. To characterize the differences between benign and malignant colorectal tissues, we have created an automated optical scattering coefficient mapping algorithm using an optical coherence tomography (OCT) system. A novel feature called the angular spectrum index quantifies the scattering coefficient distribution. In addition to scattering, subsurface morphological changes are also associated with the development of colorectal cancer. We have observed a specific mucosa structure indicating normal human colorectal tissue, and have developed a real-time pattern recognition neural network to localize this specific structure …

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

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 …

Development Of High-Speed Photoacoustic Imaging Technology And Its Applications In Biomedical Research, Yun He

McKelvey School of Engineering Theses & Dissertations

Photoacoustic (PA) tomography (PAT) is a novel imaging modality that combines the fine lateral resolution from optical imaging and the deep penetration from ultrasonic imaging, and provides rich optical-absorption–based images. PAT has been widely used in extracting structural and functional information from both ex vivo tissue samples to in vivo animals and humans with different length scales by imaging various endogenous and exogenous contrasts at the ultraviolet to infrared spectrum. For example, hemoglobin in red blood cells is of particular interest in PAT since it is one of the dominant absorbers in tissue at the visible wavelength.The main focus of …

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 …

System Characterizations And Optimized Reconstruction Methods For Novel X-Ray Imaging, Huifeng Guan

McKelvey School of Engineering Theses & Dissertations

In the past decade there have been many new emerging X-ray based imaging technologies developed for different diagnostic purposes or imaging tasks. However, there exist one or more specific problems that prevent them from being effectively or efficiently employed. In this dissertation, four different novel X-ray based imaging technologies are discussed, including propagation-based phase-contrast (PB-XPC) tomosynthesis, differential X-ray phase-contrast tomography (D-XPCT), projection-based dual-energy computed radiography (DECR), and tetrahedron beam computed tomography (TBCT). System characteristics are analyzed or optimized reconstruction methods are proposed for these imaging modalities. In the first part, we investigated the unique properties of propagation-based phase-contrast imaging technique …

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 …