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 …

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

Electrical & Systems Engineering Publications and Presentations

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

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

Electrical & Systems Engineering Publications and Presentations

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

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

Electrical & Systems Engineering Publications and Presentations

No abstract provided.

Fundamental Limits In Measuring The Anisotropic Rotational Diffusion Of Single Molecules, Weiyan Zhou, Tingting Wu, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Many biophysical techniques, such as single-molecule fluorescence correlation spectroscopy, Förster resonance energy transfer, and fluorescence anisotropy, measure the translation and rotation of biomolecules to quantify molecular processes at the nanoscale. These methods often simplify data analysis by assuming isotropic rotational diffusion, e.g., that molecules wobble within a circular cone. This simplification ignores the anisotropy present in many biological contexts that may cause molecules to exhibit different degrees of diffusion in different directions. Here, we loosen this assumption and establish a theoretical framework for describing and measuring anisotropic rotational diffusion using fluorescence imaging. We show that anisotropic wobble is directly quantified …

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 …

Optimization And Applications Of Whispering Gallery Mode Microbubble Resonator, Maxwell Adolphson

McKelvey School of Engineering Graduate Student Theses & Dissertations

Optical biosensors are frontrunners for rapid and real-time detection of analytes, particularly for low concentrations. Among them, whispering gallery mode (WGM) resonators have recently attracted a growing focus due to their robust optomechanical and optofluidic features and high sensitivity, measuring down to single binding events in small volumes. With their unique advantages and their compatibility with different sensing modalities, these sensors have the potential to become major game changers for biomedical and environmental monitoring, among many other relevant target applications. In this dissertation we focus on one geometry of whispering gallery mode resonators, the microbubble resonator. This platform is unique, …

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.

Information-Rich 6d Single-Molecule Imaging Design Using Optimization, Tingting Wu

McKelvey School of Engineering Graduate Student Theses & Dissertations

When studying biochemical processes, localizing the targets alone will not paint a complete picture. We also need techniques to probe how biomolecules interact with one another, including how molecules are organized into larger structures, how they are oriented with respect to surrounding molecules, and how local chemical parameters, like pH and hydrophobicity, vary spatially and temporally. My dissertation focuses on single-molecule orientation localization microscopy (SMOLM, Chapter 1). The objective is to develop imaging techniques for measuring how molecules are oriented with respect to their surrounding molecules, namely, 3D orientation. Achieving optimal imaging performance requires careful design of both the forward …

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 Intravital Microscopy For Brain Imaging With Improved Spatiotemporal Coverage And Contrast, Yifeng Zhou

McKelvey School of Engineering Graduate Student Theses & Dissertations

Capitalizing on the Brownian motion of red blood cells, the distinct optical absorption spectra of oxy-hemoglobin and deoxy-hemoglobin, and the blood flow-induced signal decorrelation, multi-parametric photoacoustic microscopy (PAM) is capable of quantifying concentration of hemoglobin (CHb), blood oxygen saturation (sO2), and hemodynamics at the microscopic level via statistical, spectroscopic, and correlation analyses. Multi-parametric PAM has found broad biomedical applications. In fact, there are still much room to further improve the performance and imaging capability of multi-parametric PAM to meet the ever-increasing demanding requirements of biomedical research. To date, there are two main obstacles that prevent multi-parametric PAM from being more …

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

McKelvey School of Engineering Graduate Student Theses & Dissertations

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

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

Electrical & Systems Engineering Publications and Presentations

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

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

Electrical & Systems Engineering Publications and Presentations

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

Non-Hermitian Physics And Engineering In Whispering Gallery Mode Microresonators, Changqing Wang

McKelvey School of Engineering Graduate Student Theses & Dissertations

Non-Hermitian physics describes the behaviors of open systems which have interactions with the environment. It can be applied to a wide range of classical and quantum systems. Exotic physical phenomena are unveiled in such non-Hermitian systems, especially around a singular point in the parameter space, i.e., the exceptional point (EP), where the eigenvalues and the associated eigenvectors are degenerate. A plethora of demonstrations have been found in optics and photonics, where the non-Hermitian effects are ubiquitous due to the existence of optical dissipation or amplification. In particular, whispering gallery mode (WGM) resonators are ideal candidates for studying light-matter interactions in …

Nonlinear Effects In Whispering Gallery Mode Microresonators And Their Applications, Yihang Li

McKelvey School of Engineering Graduate Student Theses & Dissertations

In a nonlinear optical system, the output optical fields are not linearly related to the power of input optical fields. On the contrary, the input fields could modify the optical properties of the system comprehensively, inducing a change in the transmission/reflection spectrum or the generation of light at different wavelengths. However, such nonlinear phenomena often require enormous optical pump power to be observed. In Whispering Gallery Mode (WGM) microresonators with high quality (Q) factors and small mode volumes (V), an intense intra-cavity optical intensity could be achieved by the spatial confinement and the temporal accumulation of the input light. Thus, …

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

Mutual Interaction Induced Multi-Particle Physics In Qed Systems – Cooperative Spontaneous Emission And Photonic Dimer Enhanced Two-Photon Excitation, Yao Zhou

McKelvey School of Engineering Graduate Student Theses & Dissertations

In recent years, the study of quantum electrodynamics (QED) in light-matter interactions has discovered various interesting phenomenons that orient many applications. However, due to the ambient entanglement among photons and atoms, few-particle dynamics remains challenging to analyze precisely and limits the progress in several fields. In few-particle systems, different number of atoms interacting with the light field generates drastically different results, even when there is only a single photon involved in the system. The interference between individual atom’s spontaneous emission wavefunctions can cooperatively alter the effective atom-light coupling strength. Depending on the spatial distance between individual of atoms and 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 …

Biomedical Applications Of Polarimetry, Nathaniel Owen King

McKelvey School of Engineering Graduate Student Theses & Dissertations

Non- or minimally-invasive data collection is highly desirable for gathering the broad base of information that facilitates moving technology to the point of care. Polarimetry has been making this transition for years. Early forms of polarization-sensitive optics involved custom multiplexing a series of images to generate a single image, but today’s snapshot polarimeters are readily commercially available.

The research presented in this dissertation advances three areas of polarimetric imaging: First, this research develops a reflectance-based measurement for tracking changes in the alignment of dynamically loaded soft tissue, specifically tendon. This work highlights a range of mounting angles across which signal …

Structural Organization And Chemical Activity Revealed By New Developments In Single-Molecule Fluorescence And Orientation Imaging, Tianben Ding

McKelvey School of Engineering Graduate Student Theses & Dissertations

Single-molecule (SM) fluorescence and its localization are important and versatile tools for understanding and quantifying dynamical nanoscale behavior of nanoparticles and biological systems. By actively controlling the concentration of fluorescent molecules and precisely localizing individual single molecules, it is possible to overcome the classical diffraction limit and achieve 'super-resolution' with image resolution on the order of 10 nanometers.

Single molecules also can be considered as nanoscale sensors since their fluorescence changes in response to their local nanoenvironment. This dissertation discusses extending this SM approach to resolve heterogeneity and dynamics of nanoscale materials and biophysical structures by using positions and orientations …

Quantitative Fluorescence Imaging For Investigation And Treatment Of Disease, Lemoyne Michael Habimana-Griffin

McKelvey School of Engineering Graduate Student Theses & Dissertations

Molecular imaging is a powerful tool that enables interrogation of basic molecular mechanisms, diagnosis of disease, guidance of therapeutic modalities and monitoring of treatment response. Among the various imaging modalities, optical imaging is particularly suited for preclinical molecular imaging owing to its high sensitivity, lack of exposure to ionizing radiation, low cost, portability and scalability of imaging from the microscopic to macroscopic scale. In particular, fluorescence molecular tomography (FMT) provides quantitative 3D reconstructions of fluorescence distributions down to picomole quantities allowing for whole animal molecular imaging. In this work, FMT is applied to detect disease-specific molecular probes, to monitor and …

A Computationally-Efficient Bound For The Variance Of Measuring The Orientation Of Single Molecules, Tingting Wu, Tianben Ding, Hesam Mazidi, Oumeng Zhang, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Modulating the polarization of excitation light, resolving the polarization of emitted fluorescence, and point spread function (PSF) engineering have been widely leveraged for measuring the orientation of single molecules. Typically, the performance of these techniques is optimized and quantified using the Cramér-Rao bound (CRB), which describes the best possible measurement variance of an unbiased estimator. However, CRB is a local measure and requires exhaustive sampling across the measurement space to fully characterize measurement precision. We develop a global variance upper bound (VUB) for fast quantification and comparison of orientation measurement techniques. Our VUB tightly bounds the diagonal elements of the …

Measuring Localization Confidence For Quantifying Accuracy And Heterogeneity In Single-Molecule Super-Resolution Microscopy, Hesam Mazidi, Tianben Ding, Arye Nehorai, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

We present a computational method, termed Wasserstein-induced flux (WIF), to robustly quantify the accuracy of individual localizations within a single-molecule localization microscopy (SMLM) dataset without ground- truth knowledge of the sample. WIF relies on the observation that accurate localizations are stable with respect to an arbitrary computational perturbation. Inspired by optimal transport theory, we measure the stability of individual localizations and develop an efficient optimization algorithm to compute WIF. We demonstrate the advantage of WIF in accurately quantifying imaging artifacts in high-density reconstruction of a tubulin network. WIF represents an advance in quantifying systematic errors with unknown and complex distributions, …

Nanoscale Colocalization Of Fluorogenic Probes Reveals The Role Of Oxygen Vacancies In The Photocatalytic Activity Of Tungsten Oxide Nanowires, Meikun Shen, Tianben Ding, Steven T. Hartman, Fudong Wang, Christina Krucylak, Zheyu Wang, Che Tan, Bo Yin, Rohan Mishra, Matthew D. Lew, Bryce Sadtler

Electrical & Systems Engineering Publications and Presentations

Defect engineering is a strategy that has been widely used to design active semiconductor photocatalysts. However, understanding the role of defects, such as oxygen vacancies, in controlling photocatalytic activity remains a challenge. Here, we report the use of chemically triggered fluorogenic probes to study the spatial distribution of active regions in individual tungsten oxide nanowires using super-resolution fluorescence microscopy. The nanowires show significant heterogeneity along their lengths for the photocatalytic generation of hydroxyl radicals. Through quantitative, coordinate-based colocalization of multiple probe molecules activated by the same nanowires, we demonstrate that the nanoscale regions most active for the photocatalytic generation of …

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

McKelvey School of Engineering Graduate Student 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 …

Polarization Division Multiplexing For Optical Data Communications, Darko Ivanovich

McKelvey School of Engineering Graduate Student Theses & Dissertations

Multiple parallel channels are ubiquitous in optical communications, with spatial division multiplexing (separate physical paths) and wavelength division multiplexing (separate optical wavelengths) being the most common forms. In this research work, we investigate the viability of polarization division multiplexing, the separation of distinct parallel optical communication channels through the polarization properties of light. We investigate polarization division multiplexing based optical communication systems in five distinct parts. In the first part of the work, we define a simulation model of two or more linearly polarized optical signals (at different polarization angles) that are transmitted through a common medium (e.g., air), filtered …

Erratum: "Imaging The Three‐Dimensional Orientation And Rotational Mobility Of Fluorescent Emitters Using The Tri‐Spot Point Spread Function", Oumeng Zhang, Jin Lu, Tianben Ding, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

In the original paper, a calibration error exists in the image-formation model used to analyze experimental images taken by our microscope, causing a bias in the orientation measurements in Figs. 2 and 3. The updated measurements are shown in Fig. E1. We have also updated the supplementary material for the original article to discuss the revised PSF model and estimation algorithms (supplementary material 2) and show the revised model and measurements (Figs. S1, S3, S7, S8, and S10–S13).