Physics 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 …

Design & Analysis Of Mixed-Mode Integrated Circuit For Pulse-Shape Discrimination, Bryan Orabutt

McKelvey School of Engineering Theses & Dissertations

In nuclear science experiments it is usually necessary to determine the type of radiation, its energy and direction with considerable accuracy. The detection of neutrons and discriminating them from gamma rays is particularly difficult. A popular method of doing so is to measure characteristics intrinsic to the pulse shape of each radiation type in order to perform pulse-shape discrimination (PSD).

Historically, PSD capable systems have been designed with two approaches in mind: specialized analog circuitry, or digital signal processing (DSP). In this work we propose a PSD capable circuit topology using techniques from both the analog and DSP domains. We …

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 …

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 …

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

McKelvey School of Engineering 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 …

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, …

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 …

Polarization Division Multiplexing For Optical Data Communications, Darko Ivanovich

McKelvey School of Engineering 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).

Fundamental Limits Of Measuring Single-Molecule Rotational Mobility, Oumeng Zhang, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

Various methods exist for measuring molecular orientation, thereby providing insight into biochemical activities at nanoscale. Since fluorescence intensity and not electric field is detected, these methods are limited to measuring even-order moments of molecular orientation. However, any measurement noise, for example photon shot noise, will result in nonzero measurements of any of these even-order moments, thereby causing rotationally-free molecules to appear to be partially constrained. Here, we build a model to quantify measurement errors in rotational mobility. Our theoretical framework enables scientists to choose the optimal single-molecule orientation measurement technique for any desired measurement accuracy and photon budget.

Instrumentation For Cryogenic Dynamic Nuclear Polarization And Electron Decoupling In Rotating Solids, Faith Joellen Scott

Arts & Sciences Electronic Theses and Dissertations

Dynamic nuclear polarization (DNP) increases the sensitivity of nuclear magnetic resonance (NMR) using the higher polarization of electron radical spins compared to nuclear spins. The addition of electron radicals for DNP to the sample can cause hyperfine broadening, which decreases the resolution of the NMR resonances due to hyperfine interactions between electron and nuclear spins. Electron decoupling has been shown to attenuate the effects of hyperfine coupling in rotating solids. Magic angle spinning (MAS) DNP with electron decoupling requires a high electron Rabi frequency provided by a high-power microwave source such as a frequency-agile gyrotron. This dissertation describes the development …

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

Fluorescence photons emitted by single molecules contain rich information regarding their rotational motions, but adapting single-molecule localization microscopy (SMLM) to measure their orientations and rotational mobilities with high precision remains a challenge. Inspired by dipole radiation patterns, we design and implement a Tri-spot point spread function (PSF) that simultaneously measures the three-dimensional orientation and the rotational mobility of dipole-like emitters across a large field of view. We show that the orientation measurements done using the Tri-spot PSF are sufficiently accurate to correct the anisotropy-based localization bias, from 30 nm to 7 nm, in SMLM. We further characterize the emission anisotropy …

Measuring 3d Molecular Orientation And Rotational Mobility Using A Tri-Spot Point Spread Function, Oumeng Zhang, Tianben Ding, Jin Lu, Hesam Mazidi, Matthew D. Lew

Electrical & Systems Engineering Publications and Presentations

We present a method to measure the molecular orientation and rotational mobility of single-molecule emitters by designing and implementing a Tri-spot point spread function. It can measure all degrees of freedom related to molecular orientation and rotational mobility. Its design is optimized by maximizing the theoretical limit of its measurement precision. We evaluate the precision and accuracy of the Tri-spot PSF by measuring the orientation and effective rotational mobility of single fluorescent molecules embedded in a polymer matrix.

Goggle Augmented Imaging And Navigation System For Fluorescence-Guided Surgery, Suman Bikash Mondal

McKelvey School of Engineering Theses & Dissertations

Surgery remains the only curative option for most solid tumors. The standard-of-care usually involves tumor resection and sentinel lymph node biopsy for cancer staging. Surgeons rely on their vision and touch to distinguish healthy from cancer tissue during surgery, often leading to incomplete tumor resection that necessitates repeat surgery. Sentinel lymph node biopsy by conventional radioactive tracking exposes patients and caregivers to ionizing radiation, while blue dye tracking stains the tissue highlighting only superficial lymph nodes. Improper identification of sentinel lymph nodes may misdiagnose the stage of the cancer. Therefore there is a clinical need for accurate intraoperative tumor and …

Optical Resonators And Fiber Tapers As Transducers For Detection Of Nanoparticles And Bio-Molecules, Huzeyfe Yilmaz

McKelvey School of Engineering Theses & Dissertations

In recent years, detection of biological interactions on single molecule level has aspired many researchers to investigate several optical, chemical, electrical and mechanical sensing tools. Among these tools, toroidal optical resonators lead the way in detection of the smallest particle/molecule with the real time measurements. In this work, bio-sensing capabilities of toroidal optical resonators are investigated. Bio-sensing is realized via measuring the analyte-antigen interaction while the antigen is immobilized through a novel functionalization method.

Not long ago, detection of single nanoparticles using optical resonators has been accomplished however the need for cost-effective and practical transducers demands simpler tools. A tapered …