Optics Commons^™

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 …

Characterizing Stiffness Dynamics Of Normal And Malignant Breast Spheroids Using Brillouin Microscopy, Razanne Rafat Zaghloul, Karlin Hilai, Chenjun Shi, Jitao Zhang

Medical Student Research Symposium

Background: Breast cancer progression and metastasis are closely linked to alterations in the mechanical properties of tumor cells and their microenvironment. Softer, more deformable cells are often associated with higher metastatic potential. While atomic force microscopy (AFM) is the current gold standard for mechanical characterization, it is limited to surface measurements and can damage 3D cultures. It remains unclear how the mechanical properties evolve over time in normal versus malignant spheroids. This study utilizes Brillouin light-scattering microscopy, a non-contact and label-free optical technique, to assess stiffness changes in normal and malignant breast epithelial spheroids over time. Understanding these mechanical signatures …

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 …

Integrated Optical Probes For Confocal Scanning Imaging And Adjustable Coherent-Gated Dynamic Sensing, Yonglin Huang

Graduate Studies Theses and Dissertations 2026

Optics and photonics have been one of the most important sciences and technologies that impact modern human life in a big way. For example, fiber-optics for communications and artificial intelligence. Optical probes are critical components for optical imaging and optical sensing technologies that have been actively researched and developed in the past decades. Advanced fiber-optic sensor probes with smaller size, better performance, lower noise, higher photon efficiency, rapid sensing time, and lower cost are needed in many applications, such as nanoscale material science, chemistry, and biomedical fields, etc.  In this project, new fiber-optic sensor probe technologies and integrated micro-optic devices …

Volumetric Fluorescence Microscopy For High-Throughput And High-Sensitivity Imaging: From Single Molecules To Tissues, Le-Mei Wang

Graduate Studies Theses and Dissertations 2026

Fluorescence microscopy is an indispensable tool in the biological sciences, enabling researchers to investigate intricate subcellular structures, particularly for volumetric studies. However, conventional optical microscopy for volumetric imaging remains fundamentally constrained by imaging speed and throughput. To bypass traditional serial z-scanning, we introduce an axially scan-free method using a phase layer cake to modulate the system's point spread function. This approach projects volumetric information onto a 2D plane in a single shot, offering high flexibility in tuning axial depth alongside simultaneous multicolor imaging with high spatial resolution and sensitivity. This dissertation divides these technical advancements into cellular and tissue imaging …

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. …

Low-Cost Cutaneous Protoporphyrin Ix (Ppix) Detection (Cpd) Device For Follow-Up Monitoring Of Patients After Photodynamic Therapy, Md Asaduzzaman Rasel

Graduate Masters Theses

Background: Photodynamic Therapy (PDT) utilizes specific wavelengths of light to activate photosensitizing chemical compounds, known as photosensitizers, which induce the generation of cytotoxic reactive oxygen species (ROS) for the targeted destruction of cancer cells. Among various photosensitizers for PDT, Protoporphyrin IX (PpIX) is widely employed in oncology and dermatology due to its natural in situ generation via the metabolic conversion of 5- aminolevulinic acid (ALA), a non-phototoxic prodrug. Systemic administration of ALA after 3-6 hr drug delay leads to peak PpIX accumulation in tissues, facilitating therapeutic and diagnostic applications. However, PpIX can persist in the skin for 24–48 hours post-treatment, …

Design And Development Of A Standalone Digital Holographic Microscope Employing Phase-Driven Reconstruction And Classification For Biomedical Imaging And Optical Diagnostics, Charlotte Kyeremah

Graduate Doctoral Dissertations

Access to advanced biomedical imaging technologies remains a significant challenge in resource-limited settings, especially for early disease detection and monitoring of diseases such as malaria, HIV, and other blood-borne diseases. Although point-of-care (POC) devices have gained popularity in global health, many rely on antibody-based tests, lateral flow strips, or optical readouts that often lack quantitative capabilities, sensitivity to early infections, or versatility in different diagnostic targets. In addition, these systems are typically dependent on disposable reagents or manual interpretation, which limits their effectiveness in remote areas. Digital Holographic Microscopy (DHM) presents a promising alternative as a label-free imaging method capable …

Volumetric Imaging Of Cellular Dynamics Using Light-Sheet Microscopy: From Subcellular Structures To Whole Organisms, Md Nasful Huda Prince

Optical Science and Engineering ETDs

Advancing biomedical imaging requires platforms that combine high-resolution, speed, and stability across biological scales. This dissertation presents three innovations designed to address limitations of conventional light-sheet and oblique plane imaging. First, we introduce Reflected Inline Detection in Epi-Oblique Plane Microscopy (RIDE-OPM), a compact and drift-resistant system that eliminates a tertiary detection path while maintaining alignment flexibility, enabling robust long-term live imaging. Second, we present a high-speed Axially Swept Light-Sheet Microscopy (ASLM) platform for cleared tissues, using dual foci and synchronized sweeping to achieve isotropic sub-micron resolution at up to 40 frames-per-second, a fourfold improvement over standard ASLM. Integrated with a …

Impact Of Shear Flow On Marine Biofilms Using Hyperspectral Imaging, Shantanu Mahesh Kore

All Theses

Various microbial communities in marine biofilms cause biofouling on submerged surfaces, posing challenges to marine industries. Despite their ecological and economic importance, biofilms' biomechanical and biochemical responses to hydrodynamic shear stress are insufficiently understood, particularly in dynamic flow conditions. To fill this gap, our study uses an innovative fiber-optic hyperspectral imaging (HSI) system and a supercontinuum laser source to examine marine biofilms' structure, composition, and detachment behavior at different shear stress levels.

To detect spectral and spatial heterogeneity in live biofilms without labeling, we created a custom imaging pipeline that captures reflectance spectra in the 600-850 nm range, targeting microbial …

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

Electrical & Systems Engineering Publications and Presentations

No abstract provided.

Noncontact Diffuse Reflectance Spectroscopy Of Synovial Fluid Samples For Rapid Identification Of Infections, Erin E. Drewke, Robert L. Brand, Caroline G. Geels, Hanna K. Jensen, Kevin Wong, Jarret D. Sanders, Narasimhan Rajaram

Biomedical Engineering Faculty Publications and Presentations

Severe joint infections, such as septic arthritis, require rapid diagnostic testing of the synovial fluid aspirated from joints level so that a surgical team can be assembled quickly. We present a diffuse reflectance spectroscopy (DRS) system for noncontact determination of infection. Using a light-tight syringe holder and fiber optic probe, diffusely reflected light from 475 to 655 nm was acquired from 18 patient samples through the wall of a syringe in a noncontact and sterile manner. We determined the reflectance ratios at two different wavelengths—R ₄₉₀/R ₆₀₀ and R ₅₈₀/R ₆₀₀ and found statistically significant …

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 …

Computational Microscopy For Biomedical Imaging With Deep Learning Assisted Image Analysis, Yuwei Liu

Dissertations

Microscopy plays a crucial role across various scientific fields by enabling structural and functional imaging with microscopic resolution. In biomedicine, microscopy contributes to basic research and clinical diagnosis. Conventionally, optical microscopy derives its contrast from the amplitude of the optical wave and provides visualization of the physical structure of the sample qualitatively. To understand the function at the cellular or tissue level, there is a need to characterize the sample quantitatively and explore contrast mechanisms other than light intensity. Image enhancement or reconstruction from microscopic imaging systems is known as computational microscopy, and it involves the application of computational techniques …

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.

In Vivo Measurement Of Nadh Fluorescence Lifetime In Skeletal Muscle Via Fiber-Coupled Time-Correlated Single Photon Counting, Kathryn M. Priest, Jacob V. Schluns, Nathania Nischal, Colton L. Gattis, Jeffery C. Wolchok, Timothy J. Muldoon

Biomedical Engineering Faculty Publications and Presentations

Nicotinamide adenine dinucleotide (NADH) is a cofactor that serves to shuttle electrons during metabolic processes such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS). NADH is autofluorescent, and its fluorescence lifetime can be used to infer metabolic dynamics in living cells. Fiber-coupled time-correlated single photon counting (TCSPC) equipped with an implantable needle probe can be used to measure NADH lifetime in vivo, enabling investigation of changing metabolic demand during muscle contraction or tissue regeneration. This study illustrates a proof of concept for point-based, minimally-invasive NADH fluorescence lifetime measurement in vivo. Volumetric muscle loss (VML) injuries were …

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 …

Frequency Domain Diffuse Optics Spectroscopies For Quantitative Measurement Of Tissue Optical Properties, Sadhu Moka

USF Tampa Graduate Theses and Dissertations

Tissue oxygen saturation, blood flow and blood volume are physiological bio-markers of tissue health. Diffuse Optical Spectroscopy(DOS) and Diffuse Correlation Spectroscopy (DCS) are two complementary approaches to measure tissue oxygen saturation and blood flow respectively. Quantitative Diffuse Optical Spectroscopy (DOS) uses multi-spectral intensities of near-infrared light that have been modulated at RF frequencies to estimate static tissue optical properties and hence concentrations of oxygenated and de-oxygenated hemoglobin. Diffuse Correlation Spectroscopy estimates tissue dynamics - i.e., blood flow, by measuring temporal intensity auto-correlation function of backscattered light diffusing through the tissue. Conventionally, DCS instruments use coherent light sources with constant intensity. …

Reducing Instrumentation Barriers Of Diffuse Correlation Spectroscopy For Low-Cost Deep Tissue Blood Flow Monitoring, Arindam Biswas

USF Tampa Graduate Theses and Dissertations

Cerebral blood flow (CBF) is a good indicator of brain health as blood carries necessary nutrients, oxygen, and metabolic byproducts. Quantitative blood flow information can be used in several clinical and therapeutic applications such as stroke detection, measuring autoregulation, evaluating brain injury, or determining neuronal activity. Over the past few decades, light-based deep tissue hemodynamic detection modalities have become popular for non-invasive CBF measurements. In particular, noninvasive Diffuse Correlation Spectroscopy (DCS), has become a tool of choice for research and clinical applications due to its depth sensitivity (>1 cm), portability, validity against other technologies such as Magnetic Resonance Imaging …

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 …

Porous Silicon Photonics For Label-Free Interferometric Biosensing And Flat Optics, Tahmid Hassan Talukdar

All Dissertations

This dissertation uses porous silicon as a material platform to explore novel optical effects in three domains: (i) It studies dispersion engineering in integrated waveguides to achieve high performance group index sensing. With proper design parameters, the sensor waveguides can theoretically achieve 6 times larger group index shift compared to the actual bulk effective refractive index shift. We demonstrate the guided mode confinement factor to be a key parameter in design and implementation of these waveguides. (ii) It explores multicolor laser illumination to experimentally demonstrate perceptually enhanced colorimetric sensing, overcoming the limitations faced by many contemporary colorimetric sensors. Our technique …

Finding Signal In The Noise: High-Fidelity, Quantitative, Optical Blood Perfusion Imaging With Interference, Abdul Mohaimen Safi

USF Tampa Graduate Theses and Dissertations

For label-free, non-invasive, wide field-of-view (FOV) imaging/monitoring of blood flow, speckle-based approaches are gaining popularity. However, to obtain quantitative flow information, speckle techniques rely on the multi-exposure scheme which requires complex, bulky, and expensive instrumentation, limiting its application to preclinical studies. This dissertation directly addresses these issues. In the first part of this dissertation, we report a novel single shot synthetic multi-exposure speckle imaging (syMESI) method to synthetically produce multi-exposure images from one short single exposure speckle image using spatial binning/averaging. We demonstrate that syMESI can reimagine conventional hardware based MESI, with low-cost single exposure laser speckle imaging (LSCI) instrumentation. …

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 …

The Aquatic Particle Number Quandry, Alexander B. Bochdansky, Huanqing Huang, Maureen H. Conte

OES Faculty Publications

Optical surveys of aquatic particles and their particle size spectra have become important tools in studies of light propagation in water, classification of water masses, and the dynamics of trophic interactions affecting particle aggregation and flux. Here, we demonstrate that typical settings used in image analysis vastly underestimate particle numbers due to the particle – gel continuum. Applying a wide range of threshold values to change the sensitivity of our detection system, we show that macrogels cannot be separated from more dense particles, and that a true particle number per volume cannot be ascertained; only relative numbers in relation 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 Graduate Student 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 …