Physical Sciences and Mathematics 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.

The Use Of Fluorescence Lifetime Imaging (Flim) For In Situ Microbial Detection In Complex Mineral Substrates, Yekaterina G. Chmykh, Jay Nadeau

Physics Faculty Publications and Presentations

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM …

An Ngqd Based Diagnostic Tool For Pancreatic Cancer, Ryan Ketan Ajgaonkar, Bong Lee, Alina Valimukhametova, Anton Naumov, Giridhar Akkaraju

Research Symposium

Background: Pancreatic cancer remains difficult to detect at early stages which contributes to a poor five-yearsurvival rate. Therefore, early detection approaches based on novel technologies should be explored to address this critical health issue. Nanomaterials have recently emerged as frontrunners for diagnostic applications due to their small size in the 1-100 nm range, which facilitates one-on-one interactions with a variety of biomolecules like oligonucleotides and makes them suitable for a plethora of detection and delivery applications. In this work, the presence of specific pancreatic cancer miRNA (pre-miR-132) is detected utilizing the fluorescence properties of highly biocompatible nitrogen-doped graphene quantum dots …

Effect Of Graphene Oxide On Spectral-Luminescenct Properties Of Xanthene Dye, E. V. Seliverstova, E. Zh. Alikhaidarova, N. Kh. Ibrayev

Eurasian Journal of Physics and Functional Materials

The effect of graphene oxide on the spectral and luminescent properties of xanthene dyes was studied. During the interaction of graphene oxide (GO) with a cationic dye, aggregation of Rhodamine 6G occurs, which is expressed as an increase in the optical density of the short-wavelength shoulder in the absorption spectrum of the dye. GO sheets can act as adsorption centers for dye molecules due to the influence of the Coulomb and intermolecular forces between GO and Rhodamine 6G. This effect is practically not pronounced for anionic dyes. The quenching of the dye fluorescence intensity in the presence of GO was …

Investigation Of G Protein-Coupled Receptor Quaternary Structure Through Fluorescence Micro-Spectroscopy And Theoretical Modeling: Interdependence Between Receptor-Receptor And Receptor-Ligand Interactions, Joel David Paprocki

Theses and Dissertations

Proteins are of high interest in biophysics research due to the important roles they play within cells, such as sensing of chemical (ions and small molecules) and physical (e.g., light) stimuli, providing structure, transporting ions/molecules, signaling, and intercellular communication. The studies described in this dissertation focus on a particular type of membrane proteins known as G protein-coupled receptors (GPCR), which play a key role in cellular response to external stimuli. We used the sterile 2 α-factor mating pheromone receptor (Ste2), a prototypical class D GPCR present within Saccharomyces cerevisiae (baker’s yeast). Ste2 is responsible for initiating the second messenger signal …

Investigatin Actin-Myosin Mechanics To Model Heart Disease Using Fluorescence Microscopy And Optical Trapping, Justin Edward Reynolds

Honors Theses

Hypertrophic cardiomyopathy (HCM) is a hereditary disease in which the myocardium becomes hypertrophied, making it more difficult for the heart to pump blood. HCM is commonly caused by a mutation in the β-cardiac myosin II heavy chain. Myosin is a motor protein that facilitates muscle contraction by converting chemical energy from ATP hydrolysis into mechanical work and concomitantly moving along actin filaments. Optical tweezers have been used previously to analyze single myosin biophysical properties; however, myosin does not work as a single unit within the heart. Multiple myosin interacts to displace actin filaments and do not have the same properties …

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 …

Spectroscopic Study On Pseudomonas Aeruginosa Biofilm In The Presence Of The Aptamer-Dna Scaffolded Silver Nanoclusters, Bidisha Sengupta, Prakash Adhikari, Esther Mallet, Ronald Havner, Prabhakar Pradhan

Faculty Publications

We report the effectiveness of silver nanocluster (Ag-NC) against the biofilm of Pseudomonas aeruginosa (PA). Two DNA aptamers specific for PA and part of their sequences were chosen as templates for growing the Ag-NC. While circular dichroism (CD) studies determined the presence of secondary structures, UV/Vis absorption, and fluorescence spectroscopic studies confirmed the formation of the fluorescent Ag-NC on the DNA templates. Furthermore, mesoscopic physics-based partial wave spectroscopy (PWS) was used to analyze the backscattered light signal that can detect the degree of nanoscale mass density/refractive index fluctuations to identify the biofilm formation, comparatively among the different aptamers with respect …

Gold/Qds-Embedded-Ceria Nanoparticles: Optical Fluorescence Enhancement As A Quenching Sensor, Nader Shehata, Effat Samir, Ishac Kandas

Electrical & Computer Engineering Faculty Publications

This work focuses on improving the fluorescence intensity of cerium oxide (ceria) nanoparticles (NPs) through added plasmonic nanostructures. Ceria nanoparticles are fluorescent nanostructures which can emit visible fluorescence emissions under violet excitation. Here, we investigated different added plasmonic nanostructures, such as gold nanoparticles (Au NPs) and Cadmium sulfide/selenide quantum dots (CdS/CdSe QDs), to check the enhancement of fluorescence intensity emissions caused by ceria NPs. Different plasmonic resonances of both aforementioned nanostructures have been selected to develop optical coupling with both fluorescence excitation and emission wavelengths of ceria. In addition, different additions whether in-situ or post-synthesis have been investigated. We found …

Investigating Ions’ Effects On The Fluorescent Protein Dendra2, Benjamin Waterman

Honors College

While superresolution microscopy has opened the doors to insights into biological phenomena we couldn’t have dreamed of in the last century, its methodology is naturally limited. We aim to push the envelope of its capabilities by testing the effect that Ca2+ and H+ ions have on the fluorescent protein Dendra2. Utilizing a newly designed perfusion chamber, we flow separate solutions containing Ca2+ and H+ ions into a cellular environment, in which the cells in question have been tagged with Dendra2. Utilizing the superresolution technique known as Spectral Fluorescence Photoactivation Localization Microscopy, we are able to obtain information about the emission …

Decreasing Of The Concentration Quenching Of Fluorescent Dye In A Solution By Introducing It To Nanoparticles Of Silicon Dioxide, A. I. Mantel, A. E. Yensebayeva, S. B. Smatov, I. S. Irgibayeva

Eurasian Journal of Physics and Functional Materials

The technique of introduction a fluorescent dye Tris(bipyridine)ruthenium (II) chloride into nanoparticles of silicon dioxide in isopropyl alcohol was tested. The preparation of nanoparticles was carried out by means of the Stober method. The optimal reaction conditions were determined. It was found out that the penetration of a dye into silicon dioxide nanoparticles reduced the effect of concentration quenching of the dye and made it possible to achieve an increase in fluorescence intensity in the solution more than twofold. An attempt was made to functionalize the obtained particles by introducing amino groups onto the particle surface. Fused silver nanoparticles were …

High-Throughput Automated Multi-Target Super-Resolution Imaging, Farzin Farzam

Physics & Astronomy ETDs

Super-resolution microscopy techniques developed through the past few decades enable us to surpass the classical diffraction limit of light, and thus open new doors to investigate the formerly inaccessible world of nanometer-sized objects. Most importantly, by using super-resolution microscopy, one can visualize sub-cellular structures in the range of 10 to 200 nm. At this range, we can investigate exciting problems in biology and medicine by visualizing protein-protein interactions and spatiotemporal analysis of structures of interest on the surface or inside cells. These techniques (collectively known as nanoscopy) have a high impact on understanding and solving biological questions. This dissertation starts …

Two-Photon Excitation Of Cesium Alkali Metal Vapor 72D, 82D Kinetics And Spectroscopy, Ricardo C. Davila

Theses and Dissertations

Pulsed excitation on the two-photon Cs 6²S_½ → 7²D_3/2,5/2 transition results in time-resolved fluorescence at 697 nm and 672 nm. The rates for fine structure mixing between the 7²D_3/2,5/2 states have been measured for helium and argon rare gas collision partners. The mixing rates are very fast, 1.26 ± 0.05 x 10^-9 cm³/(atom sec) for He and 1.52 ± 0.05 x 10^-10 cm³ /(atom sec) for Ar, driven by the small energy splitting and large radial distribution for the valence electron. …

Influence Of Overlapping Between The Fluorescence Spectra Of Dye Molecules And The Spectrum Of Plasmon Absorption Of Silver Nanoparticles On The Luminescence Of Laser Dyes In Ethyl Alcohol, A. K. Aimukhanov, N. Kh. Ibrayev

Eurasian Journal of Physics and Functional Materials

In the present work, the influence of the degree of overlapping between the plasmon absorption spectrum of silver nanoparticles and the luminescence spectra of P6Zh, phenalamine 160, and oxazine 17 on their luminescence was studied. With the addition of Ag NPs to the ethanol solution, the absorption of dyes depends on the concentration of Ag nanoparticles. The overlapping integrals between the plasmon absorption spectrum of Ag NPs and the absorption spectra of the studied dyes were 24.2 for P6J, 52 for phenalemine 160 and 38.5 for oxazine 17, respectively. The optical density in the maximum increased 1.6-fold for P6Zh, 1.4-fold …

A Study In Cross-Beam Polarization Fluorescence Photoactivation Localization Microscopy On Dendra2-Hemagluttinin In Fixed Nih3t3 Cells, Matthew M. Valles

Electronic Theses and Dissertations

Fluorescence microscopy is popular for its noninvasive properties and its use in imaging multiple species, simultaneously. Furthermore, superresolution fluorescence microscopy (SRFLM) utilizes photoswitchable proteins to improve the lateral resolution of conventional fluorescence microscopy by an order of magnitude. There is little work conducted on the study of excitation laser polarizations and their effect on the number of localizations as well as the brightness of molecules. This thesis attempts to study the effect of excitation wavelength polarization on the number of localizations and the brightness of molecules by comparing two orientations of circularly-polarized, excitation lasers. The first type of orientation involves …

Thermal Property Measurement Of Thin Fibers By Complementary Methods, Troy Robert Munro

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Thin fibers are prevalent in many engineering materials. Measuring how well heat transfers in such small fibers can be difficult to determine, and previous methods have led to erroneous results. This dissertation details three proposed methods to improve the measurement of the thermal conductivity, thermal diffusivity, and volumetric heat capacity of thin fibers. Of particular interest is natural and synthetic spider silks because previously published values of the natural silk thermal conductivity was similar to copper, an excellent thermal conductor.

The three methods developed are the improved transient electrothermal technique (which was redeveloped to include radiation and convection heat losses …

Wavelength Dependent Specific Plasmon Resonance Coupling Of Single Silver Nanoparticles With Egfp, Kerry J. Lee, Tao Huang, Prakash D. Nallathamby

Chemistry & Biochemistry Faculty Publications

Noble metal nanoparticles (NPs) possess unique plasmonic properties, enabling them to serve as sub-diffraction light sources and nano-antennae for a wide range of applications. Here we report the specific interaction of single Ag NPs with single EGFP molecules and a high dependence of their interaction upon localized-surface-plasmon-resonance (LSPR) spectra of single Ag NPs and EGFP. The LSPR spectra of single red Ag NPs show a stunning 60 nm blue-shift during their incubation with EGFP, whereas they remain unchanged during their incubation with bovine serum albumin (BSA). Interestingly, the peak wavelengths of the LSPR spectra of green and blue Ag NPs …

Near-Infrared Surface-Enhanced Fluorescence Using Silver Nanoparticles In Solution, Michael D. Furtaw

Department of Mechanical and Materials Engineering: Dissertations, Theses, and Student Research

Fluorescence spectroscopy is a widely used detection technology in many research and clinical assays. Further improvement to assay sensitivity may enable earlier diagnosis of disease, novel biomarker discovery, and ultimately, improved outcomes of clinical care along with reduction in costs. Near-infrared, surface-enhanced fluorescence (NIR-SEF) is a promising approach to improve assay sensitivity via simultaneous increase in signal with a reduction in background. This dissertation describes research conducted with the overall goal to determine the extent to which fluorescence in solution may be enhanced by altering specific variables involved in the formation of plasmonactive nanostructures of dye-labeled protein and silver nanoparticles …

Pressure Induced Structural Changes And Gas Diffusion Pathways In Monomeric Fluorescent Proteins, Yuba R. Bhandari

FIU Electronic Theses and Dissertations

Fluorescent proteins (FPs) are extremely valuable biochemical markers which have found a wide range of applications in cellular and molecular biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for in vivo applications in mammalian cell imaging. Fluorescent proteins consist of a chromophore caged in the beta-barrel protein scaffold. The photophysical properties of an FP is determined by its chromophore structure and its interactions with the protein barrel.

Application of hydrostatic pressure on FPs results in the modification of the chromophore environment which allows a systematic study of the role of the …

Cavity Modes And Their Excitations In Elliptical Plasmonic Patch Nanoantennas, Ayan Chakrabarty, Feng Wnag, Fred Minkowski, Kai Sun, Qi-Huo Wei

Qi-Huo Wei

We present experimental and theoretical studies of two dimensional periodic arrays of elliptical plasmonic patch nanoantennas. Experimental and simulation results demonstrate that the azimuthal symmetry breaking of the metal patches leads to the occurrence of even and odd resonant cavity modes and the excitation geometries dependent on their modal symmetries. We show that the cavity modes can be described by the product of radial and angular Mathieu functions with excellent agreements with both simulations and experiments. The effects of the patch periodicity on the excitation of the surface plasmon and its coupling with the cavity modes are also discussed.

Analysis Of Human Skin Tissue By Raman Microspectroscopy: Dealing With The Background, Franck Bonnier, Syed Mehmood Ali, Peter Knief, Helen Lambkin, Kathleen Flynn, Vincent Mcdonagh, Claragh Healy, Thomas Lee, Fiona Lyng, Hugh Byrne

Articles

Raman microspectroscopy is widely used for molecular characterisation of tissue samples. Nevertheless, when working in vitro on tissue sections, the presence of a broad background to the spectra remains problematic and its removal requires advanced methods for pre-processing of the data. To date, research efforts have been primarily devoted to development of techniques of statistical analysis to extract the relevant information contained in the spectra. However, few attempts have been made to understand the origin of the background and to improve the protocols used for the collection of Raman spectra that could lead to the reduction or elimination of the …

Acyl-Chain Mismatch Driven Superlattice Arrangements In Dppc/Dlpc/Cholesterol Bilayers, Brian Cannon, Anthony Lewis, Pentti Somerharju, Jorma Virtanen, Juyang Huang, Kwan H. Cheng

Physics and Astronomy Faculty Research

Fluorescence and infrared spectroscopy and cholesterol oxidase activity were employed to investigate the effect of phosphatidylcholine (PC) acyl chain length mismatch on the lateral organizations of lipids in liquid-ordered dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/CHOL) bilayers. Plots of steady-state fluorescence emission anisotropy of diphenylhexatriene (DPH) labeled PC (DPH-PC) embedded in the DPPC/DLPC/CHOL bilayers revealed significant peaks at several DPPC mole fractions (Y_DPPC) when the cholesterol mole fraction (X_CHOL) was fixed to particular values. Analogously, the DPH-PC anisotropy peaked at several critical X_CHOL’s when Y_DPPC was fixed. Acyl chain C−H and C═O vibrational peak frequencies of …

Far-Field Optical Nanoscopy Based On Continuous Wave Laser Stimulated Emission Depletion, C. Kuang, Wei Zhao, Guiren Wang

Faculty Publications

Stimulated emission depletion (STED) microscopy is one of the breakthrough technologies that belong to far-field optical microscopy and can achieve nanoscale spatial resolution. We demonstrate a far-field optical nanoscopy based on continuous wave lasers with different wavelengths, i.e., violet and green lasers for excitation and STED, respectively. Fluorescent dyes Coumarin 102 and Atto 390 are used for validating the depletion efficiency. Fluorescent nanoparticles are selected for characterizing the spatial resolution of the STED system. Linear scanning of the laser beams of the STED system along one line of a microscope slide, which is coated with the nanoparticles, indicates that a …

The Mechanical Properties Of Individual, Electrospun Fibrinogen Fibers, Christine C. Helms, Corentin Coulais, Manoj Namboothiry, David L. Carroll, Roy R. Hantgan, Martin Guthold

Physics Faculty Publications

We used a combined atomic force microscope (AFM)/fluorescence microscope technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 μm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22·10⁶ Pa. We collected incremental stress-strain curves to determine the …

Algorithm For The Analysis Of Tryptophan Flourescence Spectra And Their Correlation With Protein Structural Parameters, John Hixon, Yana K. Reshetnyak

Physics Faculty Publications

The fluorescence properties of tryptophan residues are sensitive to the microenvironment of fluorophores in proteins. Therefore, fluorescence characteristics are widely used to study structural transitions in proteins. However, the decoding of the structural information from spectroscopic data is challenging. Here we present a review of approaches developed for the decomposition of multi-component protein tryptophan fluorescence spectra and correlation of these spectral parameters with protein structural properties.

Recombination Fluorescence In Ultracold Neutral Plasmas, Scott D. Bergeson, F. Robicheaux

Faculty Publications

We present the first measurements and simulations of recombination fluorescence from ultracold neutral calcium plasmas. This method probes three-body recombination at times less than 1 µs, shorter than previously published time scales. For the lowest initial electron temperatures, the recombination rate scales with the density as n22, significantly slower than the predicted n3. Recombination fluorescence opens a new diagnostic window in ultracold plasmas. In most cases it probes deeply bound level populations that depend critically on electron energetics. However, a perturbation in the calcium 4snd Rydberg series allows our fluorescence measurements to probe the population in weakly bound levels that …

A Novel Setup For High-Pressure Raman Spectroscopy Under A Microscope, Thomas Andrew Oakeson

Electronic Theses and Dissertations

Functional properties of biological molecules and cells are affected by environmental parameters such as temperature and pressure. While Raman spectroscopy provides an intrinsic probe of molecular structural changes, the incorporation of a microscope enables studies of minuscule amounts of biological compounds with spatial resolution on a micron scale. We have developed a novel setup which combines a Raman microscope and a high pressure cell. A micro-capillary made out of fused silica simultaneously serves as the supporting body and the optical window of the pressure cell. The cell has been tested over the pressure range from 0.1 to 4 kbar. Raman …

Mechanistic Studies On The Mononuclear ZnIi-Containing Metallo-Β-Lactamase Imis From Aeromonas Sobria, Narayan Sharma, Christine E. Hajdin, Sowmya Chandrasekar, Brian Bennett, Ke-Wu Yang, Michael W. Crowder

Physics Faculty Research and Publications

In an effort to understand the reaction mechanism of a B2 metallo-β-lactamase, steady-state and pre-steady-state kinetic and rapid freeze quench electron paramagnetic resonance (EPR) studies were conducted on ImiS and its reaction with imipenem and meropenem. pH dependence studies revealed no inflection points in the pH range of 5.0−8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Site-directed mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, …

Fluorescence Measurements Of Expanding Strongly Coupled Neutral Plasmas, E. A. Cummings, J. E. Daily, Dallin S. Durfee, Scott D. Bergeson

Faculty Publications

We report new detailed density profile measurements in expanding strongly coupled neutral calcium plasmas. Using laser-induced fluorescence techniques, we determine plasma densities in the range of 10^5 to 10^9 cm^-3 with a time resolution limit as small as 7 ns. Strong coupling in the plasma ions is inferred directly from the fluorescence signals. Evidence for strong coupling at late times is presented, confirming a recent theoretical result.

Relativistic And Radiative Corrections To The Mollow Spectrum, Jorg Evers, Ulrich D. Jentschura, Christoph H. Keitel

Physics Faculty Research & Creative Works

The incoherent inelastic part of the resonance fluorescence spectrum of a laser-driven atom is known as the Mollow spectrum [B. R. Mollow, Phys. Rev. 188, 1969 (1969)]. Starting from this level of description, we discuss theoretical foundations of high-precision spectroscopy using the resonance fluorescence light of strongly laser-driven atoms. Specifically, we evaluate the leading relativistic and radiative corrections to the Mollow spectrum, up to the relative orders of ( Z α )² and α ( Z α )² , respectively, and Bloch-Siegert shifts as well as stimulated radiative corrections involving off-resonant virtual states. Complete results are provided for …