Single Molecule Super Resolution Microscopy Study On The Precision With Which Dna Nanostructures Can Orient Fluorescent Dyes

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Single Molecule Super-resolution Microscopy Study on the Precision with which DNA Nanostructures Can Orient Fluorescent Dyes

Author : Brett Michael Ward
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (134 download)

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Book Synopsis Single Molecule Super-resolution Microscopy Study on the Precision with which DNA Nanostructures Can Orient Fluorescent Dyes by : Brett Michael Ward

Download or read book Single Molecule Super-resolution Microscopy Study on the Precision with which DNA Nanostructures Can Orient Fluorescent Dyes written by Brett Michael Ward and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: "DNA nanotechnology enables the rapid, programmable self-assembly of novel structures and devices at the nanoscale. Utilizing the simplicity of Watson-Crick base pairing, DNA nanostructures are capable of assembling a variety of nanoparticles in arbitrary configurations with relative ease. Several emerging opto-electronic systems require a high degree of control of both the position and orientation of component fluorescent molecules, and while DNA nanostructures have demonstrated these capabilities, the precision with which DNA can orient fluorescent molecules is not well understood. Determining these bounds is critical in establishing the viability of DNA nanotechnology as a method of assembling fluorescent molecular networks. In this work, using a combination of single molecule emission dipole imaging and super-resolution microscopy techniques, we correlate the orientations of fluorescent dye molecules to the orientations of their DNA substrates along five degrees of freedom. Several species of dyes were embedded within a DNA sequence using either one or two covalent tethers. These strands were incorporated directly into DNA origami structures to investigate the dependence of the location and binding architecture of the dye on the orientational precision of DNA nanostructures. Dye functionalized strands were also folded into a simpler four-arm junction, which was then immobilized on an origami structure to study the influence of the DNA substrate on dye orientation. Correlated analysis of super-resolution images of origami structures and single molecule emission dipole images from the embedded fluorescent molecule within the same structure allowed us to directly measure the relative orientations of dye molecules within DNA nanostructures. The resulting measurements revealed a moderate degree of polar angle control but a large variation in azimuthal control for the majority of structures examined. These measurements establish a single-molecule method for measurement of correlated orientations and provide a powerful approach for future studies on increasing the precision in the orientational control of fluorescent dye molecule monomers by DNA nanostructures."--Boise State University ScholarWorks.

Nanoscale Optical and Correlative Microscopies for Quantitative Characterization of DNA Nanostructures

Author : Christopher Michael Green
Publisher :
ISBN 13 :
Total Pages : 150 pages
Book Rating : 4.:/5 (114 download)

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Book Synopsis Nanoscale Optical and Correlative Microscopies for Quantitative Characterization of DNA Nanostructures by : Christopher Michael Green

Download or read book Nanoscale Optical and Correlative Microscopies for Quantitative Characterization of DNA Nanostructures written by Christopher Michael Green and published by . This book was released on 2019 with total page 150 pages. Available in PDF, EPUB and Kindle. Book excerpt: "Methods to engineer nanomaterials and devices with uniquely tailored properties are highly sought after in fields such as manufacturing, medicine, energy, and the environment. The macromolecule deoxyribonucleic acid (DNA) enables programmable self-assembly of nanostructures with near arbitrary shape and size and with unprecedented precision and accuracy. Additionally, DNA can be chemically modified to attach molecules and nanoparticles, providing a means to organize active materials into devices with unique or enhanced properties. One particularly powerful form of DNA-based self-assembly, DNA origami, provides robust structures with the potential for nanometer-scale resolution of addressable sites. DNA origami are assembled from one large DNA "scaffold" strand and many unique, short "staple" strands; each staple programmatically binds the scaffold at several distant domains, and the coordinated interactions of many staples with the scaffold act to fold the scaffold into a desired shape. The utility of DNA origami has been demonstrated through multiple applications, such as plasmonic and photonic devices, electronic device patterning, information storage, drug delivery, and biosensors. Despite the promise of DNA nanotechnology, few products have successfully translated from the laboratory to industry. Achieving high yield and high-precision synthesis of stable DNA nanostructures is one of the biggest challenges to applications of DNA nanostructures. For adoption in manufacturing, methods to measure and inspect assembled structures (i.e. metrology) are essential. Common high-resolution imaging techniques used to characterize DNA nanostructures, such as atomic force microscopy and transmission electron microscopy, cannot facilitate high-throughput characterization, and few studies have been directed towards the development of improved methods for nanoscale metrology. DNA-PAINT super-resolution microscopy enables high-resolution, multiplexed imaging of reactive sites on DNA nanostructures and offers the potential for inline optical metrology. In this work, nanoscale metrologies utilizing DNA-PAINT were developed for DNA nanostructures and applied to characterize DNA origami arrays and single site defects on DNA origami. For metrology of DNA origami arrays, an embedded, multiplexed optical super-resolution methodology was developed to characterize the periodic structure and defects of two-dimensional arrays. Images revealed the spatial arrangement of structures within the arrays, internal array defects, and grain boundaries between arrays, enabling the reconstruction of arrays from the images. The nature of the imaging technique is also highly compatible with statistical methods, enabling rapid statistical analysis of synthesis conditions. To obtain a greater understanding of DNA origami defects at the scale of individual strands, correlative super-resolution and atomic force microscopies were enabled through the development of a simple and flexibl."--Boise State University ScholarWorks.

Advancing DNA Nanotechnology Using Single Molecule Fluorescence Methodologies

Author : Amani Hariri
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (964 download)

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Book Synopsis Advancing DNA Nanotechnology Using Single Molecule Fluorescence Methodologies by : Amani Hariri

Download or read book Advancing DNA Nanotechnology Using Single Molecule Fluorescence Methodologies written by Amani Hariri and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "By taking it out of its biological role, DNA has served as a robust, reliable and programmable nanofabrication building block for the assembly of a variety of two- and three-dimensional discrete nanometer-sized structures with arbitrary shapes and designs. These molecularly addressable materials could serve as platforms for the site-specific hybridization with nanometer precision. Besides structural complexity, DNA objects evolved to comprise an intrinsic dynamic character allowing them to respond to surrounding stimuli in a predictable manner. Many researchers have previously constructed nanostructures using methods that rely on the spontaneous assembly of DNA in solution, in which case the more complex the assembly design is, the more error-prone the product becomes. This constitutes a major problem for the application of DNA-based structures in dynamic devices such as machines, motors, robots, and computers, where even small errors in assembly can drastically affect performance. Therefore, quantitative tools for analysing structures and dynamics of complex DNA nanostructures are critical. Single molecule fluorescence techniques have been transformative to the field of nanoscience and have played a leading role in providing a mature understanding and an informative feedback on the building blocks and final products of self-assembly. This thesis demonstrates how different single molecule fluorescence methodologies can be utilized to study and advance the structure, dynamics and integrity of DNA-assemblies, specifically those shaped as nanotubes. Conceptually, this research can be divided into four main parts: (i) synthesis optimization, (ii) structural characterization, (iii) monitoring dynamic structures, and (iv) studying structural dynamics. First, a solid-phase synthesis strategy and its visualization through single-molecule spectroscopy was devised to assemble DNA nanotubes in a stepwise fashion, with a full control over their size and sequence pattern. This method paves the way for the production of custom-made DNA nanotubes with fewer structural flaws than the spontaneous-assembly method. Second, single molecule photobleaching and two-color colocalization approaches were uniquely combined to provide a systematic way of assessing the polydispersity, stoichiometry and degree of defectiveness of different DNA nanotubes structures. These approaches will be of significant importance for many research groups synthesizing large supramolecular structures or studying naturally occurring ones, such as protein clusters, amyloids, etc. Third, in situ single molecule immobilization-based fluorescence microscopy was employed to introduce structural changes into DNA nanotubes by dynamically adjusting one or several of the edge lengths between the building blocks using a combination of strand displacement and loops. This is interesting for sensing applications, especially when the analyte produces large scale, detectable structural changes, and also for creating molecular muscles, capable of extension and contraction under external control. Lastly, dynamics and robustness of DNA nanotubes, reconfigured in response to site-specific deletion of DNA strands, were investigated using two color single molecule microscopy. This strategy enables to develop a better understanding of the collective structural changes within DNA structures in response to modifications in their repeat unit. Together, the different methods developed in this thesis underline the importance of single molecule techniques as powerful tools which can advance the field of DNA nanotechnology by enabling the production of well-defined high-quality objects that can meet the designer's compositional and dynamic specifications." --

Single-Molecule Science

Author : Krishnarao Appasani
Publisher : Cambridge University Press
ISBN 13 : 1108423361
Total Pages : 171 pages
Book Rating : 4.1/5 (84 download)

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Book Synopsis Single-Molecule Science by : Krishnarao Appasani

Download or read book Single-Molecule Science written by Krishnarao Appasani and published by Cambridge University Press. This book was released on 2022-05-26 with total page 171 pages. Available in PDF, EPUB and Kindle. Book excerpt: A comprehensive volume that brings together authoritative overviews of single molecule science techniques from a biological perspective.

Mechanisms in Single Molecule Fluorescence Imaging

Author : Yasser Gidi
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (119 download)

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Book Synopsis Mechanisms in Single Molecule Fluorescence Imaging by : Yasser Gidi

Download or read book Mechanisms in Single Molecule Fluorescence Imaging written by Yasser Gidi and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "Single molecule fluorescence (SMF) microscopy techniques have profoundly changed the way we can study the dynamics and structure of the microscopic and nanoscopic world. SMF methods, including cutting-edge super resolution microscopy, allow unprecedented spatiotemporal resolution and are particularly powerful for the study of heterogeneous and unsynchronized systems.In order to unravel the full potential of SMF techniques, many challenges remain to be addressed, including surface passivation and a thorough mechanistic photochemical and photophysical understanding of fluorescent dyes and their associated SMF techniques.The research presented in this thesis aims to address many of the current challenges plaguing SMF studies by providing key insights behind SMF data analysis related to single molecule Förster resonance energy transfer (FRET) studies and associated protein induced fluorescent enhancement (PIFE) studies. Our work provides a mechanistic underpinning for the photochemical and photophysical behavior of cyanine dyes, of relevance to photostabilization and photoswitching towards super resolution. These results also provide a framework for sample preparation in terms of enhanced surface quality for specific substrate immobilization. Armed with these technical improvements, we next tackled key biophysical aspects related to the mechanism and dynamics of the viral replication machinery of the Hepatitis C virus (HCV). The non-structural 5B (NS5B) polymerase of HCV has the remarkable ability to initiate synthesis de novo through a primer independent mechanism. Through our single molecule work, we report on NS5B binding, sliding, and searching for the 3’ of its single stranded RNA substrate.Overall, our work provides new insights on critical aspects of SMF techniques such as sample preparation, fluorescence signal control, and data analysis. Moreover, it provides an example of the power of SMS to study relevant biomolecular mechanisms such as the replication machinery of viruses"--

Cryogenic Super-Resolved Fluorescence Microscopy

Author : Siegfried Weisenburger
Publisher : Logos Verlag Berlin GmbH
ISBN 13 : 3832543643
Total Pages : 176 pages
Book Rating : 4.8/5 (325 download)

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Book Synopsis Cryogenic Super-Resolved Fluorescence Microscopy by : Siegfried Weisenburger

Download or read book Cryogenic Super-Resolved Fluorescence Microscopy written by Siegfried Weisenburger and published by Logos Verlag Berlin GmbH. This book was released on 2016-11-16 with total page 176 pages. Available in PDF, EPUB and Kindle. Book excerpt: The significance of super-resolved fluorescence microscopy beyond the diffraction barrier was recognized by the Nobel Prize in Chemistry in 2014. At room temperature, these techniques typically achieve a resolution on the order of twenty nanometers. They already allowed for resolving subcellular structures and organelles, and are starting to enable discoveries in neuroscience, molecular biology and other life sciences. One can dream about increasing the optical resolution by another two orders of magnitude in order to directly resolve sub-molecular structures such as constituents of molecular complexes or even protein structure itself. The aim of the present work is to accomplish exactly that. In this PhD thesis, a novel microscopy technique is presented that exploits cryogenic measurements to push optical resolution to the Angstrom level. The near atomic resolution is made possible by the substantial improvement of the molecular photostability at liquid helium temperature. This method allows one to gain structural information of proteins or other molecular complexes that might not be accessible by existing analytical methods such as X-ray crystallography, cryogenic electron microscopy or magnetic resonance spectroscopy. These results mark record optical resolution and demonstrate that optical resolution can be pushed beyond the diffraction limit by nearly one thousand times.

YOYO and POPO Dye Photophysics for Super-resolution Optical Nanoscopy

Author : Joseph R. Pyle
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (112 download)

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Book Synopsis YOYO and POPO Dye Photophysics for Super-resolution Optical Nanoscopy by : Joseph R. Pyle

Download or read book YOYO and POPO Dye Photophysics for Super-resolution Optical Nanoscopy written by Joseph R. Pyle and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Single molecule fluorescence microscopy has been used in the past two decades to improve resolution and view information unattainable from bulk studies. As a relatively new field there is still a lot of areas to explore. This dissertation focuses on single molecule studies on two groups of dyes, including the YOYO-POPO-TOTO family of DNA intercalating dyes and fluorescent polymer dots. The motivation is to understand their special properties in single-molecule microscopy. In this dissertation I study YOYO-1’s quenching mechanism, its binding and bleaching in DNA, and its production of reactive oxygen species (ROS) under light illumination.

Development of Single Molecule Optical Techniques for the Study of Recognition and Repair of DNA Damage

Author : Samantha R. Fore
Publisher :
ISBN 13 :
Total Pages : 320 pages
Book Rating : 4.:/5 (74 download)

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Book Synopsis Development of Single Molecule Optical Techniques for the Study of Recognition and Repair of DNA Damage by : Samantha R. Fore

Download or read book Development of Single Molecule Optical Techniques for the Study of Recognition and Repair of DNA Damage written by Samantha R. Fore and published by . This book was released on 2006 with total page 320 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Single-molecule Fluorescence Methods for DNA Nanostructure Assembly and Actuation

Author : Casey Platnich
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (129 download)

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Book Synopsis Single-molecule Fluorescence Methods for DNA Nanostructure Assembly and Actuation by : Casey Platnich

Download or read book Single-molecule Fluorescence Methods for DNA Nanostructure Assembly and Actuation written by Casey Platnich and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "DNA nanotechnology relies on the molecular recognition properties of DNA to produce complex architectures through self-assembly. DNA nanostructures allow scientists to organize functional materials with nanoscale precision, with applications spanning materials science, bioengineering, and physics. In addition to their remarkable performance as frameworks for other species, DNA constructs also possess intrinsic dynamic properties due to the non-covalent interactions that bring them together and show exceptional promise in the areas of controlled, personalized drug delivery and the development of biosensors.Although DNA nanostructures have become increasingly intricate and functional, the development of tools to study them has lagged. Historically, ensemble measurements have been primarily used to analyze the outputs of DNA nanotechnology, averaging the behaviour of billions of molecules within a sample to generate an output. This averaging means that the structure and performance of each individual construct is obscured, concealing malformed structures and masking properties associated with heterogeneity in size, length, and shape in self-assembled samples. For integration into engineered nanomaterials and biomedical devices, it is imperative that the DNA nanomaterials within a sample be structurally identical, with predictable and controllable behaviour. This goal requires the development of new analytical tools for the real-time observation of nanostructure assembly and actuation.This thesis describes the expansion of single-molecule fluorescence methodologies to probe the self-assembly pathways of DNA nanomaterials. These techniques monitor the self-assembly kinetics of surface-grafted DNA constructs, giving rise to a new generation of monodisperse, user-defined structures prepared using fully automated methods. The dynamic nature of these measurements also allows the single-molecule differentiation of the mechanisms of supramolecular polymerization. Exploring the fine-grained detail of self-assembly will enable the actualization of DNA nanostructures in custom delivery and sensing applications. In establishing new strategies to detect the assembly pathways of DNA architectures, this thesis provides a cohesive framework towards understanding hierarchical DNA assemblies at the single-molecule level"--

Quantitative Molecular Orientation Imaging of Biological Structures by Polarized Super-resolution Fluorescence Microscopy

Author : Haitham Ahmed Shaban Ahmed
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (948 download)

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Book Synopsis Quantitative Molecular Orientation Imaging of Biological Structures by Polarized Super-resolution Fluorescence Microscopy by : Haitham Ahmed Shaban Ahmed

Download or read book Quantitative Molecular Orientation Imaging of Biological Structures by Polarized Super-resolution Fluorescence Microscopy written by Haitham Ahmed Shaban Ahmed and published by . This book was released on 2015 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: .In this thesis we built and optimized quantitative polarized stochastic super-resolution fluorescence microscopy techniques that enabled us to image molecular orientation behaviors in static and dynamic environments at single molecule level and with nano-scale resolution. Using a scheme of stochastic read-out super resolution microscopy in combination with polarized detection, we can reconstruct fluorescence anisotropy images at a spatial resolution of 40 nm. In particular, we have been able to use the techniques to quantify the molecular orientationalorder in cellular and bio-molecular assemblies. For cellular imaging, we could quantify the ability of fluorophore labels to report molecular orientation of actin and microtubules in fixed cells. Furthermore, we used the improvements of resolution and polarization detection to study molecular order of amyloid aggregates at a nanoscopic scale. Also, we studied repair protein RAD51` s interaction with DNA by using dual color polarized fluorescence microscopy, to quantify the orientational order of DNA and RAD51 to understand the homologous recombination of DNA repair mechanism.

Super-resolution Optical Lithography with DNA.

Author : Shi Ho Kim
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (125 download)

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Book Synopsis Super-resolution Optical Lithography with DNA. by : Shi Ho Kim

Download or read book Super-resolution Optical Lithography with DNA. written by Shi Ho Kim and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Super-resolution optical microscopy offers powerful imaging methods that use sub-diffraction imaging techniques and have been employed widely to investigate subcellular biological processes. While super-resolution optical microscopy can reveal structures whose size is below the diffraction limit of visible light, it has never been developed to construct structures at an optical super-resolution. Such a method will offer efficient ways to construct nanoparticle assemblies whose properties can be tuned with a precisely designed shape and internal structure at a resolution of a few tens of nanometers (nm). Nanoparticle assemblies can be useful for various applications such as novel optoelectronic devices based on metal nanoparticles. DNA has been utilized as a building material for macro-molecular assemblies due to its programmability and reversibility based on double-strand hybridization. Previously a photolithographic technique utilizing UV-cross-linkable DNA has been reported to chemically pattern a surface in micrometer resolution. By taking advantage of super-resolution microscopy and DNA photolithography, I have developed a method with the name of Super-resolution Optical Lithography with DNA (SOLiD), with which one can construct nanoparticle assemblies below the optical diffraction limit. The method utilizes a DNA hairpin and single-molecule fluorescence resonance energy transfer (smFRET). To understand SOLiD, it is crucial to understand super-resolution microscopy techniques along with the physical and mechanical properties of a DNA hairpin that render its folding and unfolding kinetics. In this dissertation, I present my research on developing an optical super-resolution microscopy method using a DNA hairpin and smFRET and on investigating the kinetics of single-stranded DNA folding and unfolding to understand the conformational dynamics of a DNA hairpin. In Chapter 1, super-resolution optical microscopy and FRET are reviewed. In Chapter 2, single-molecule fluorescent microscopy and the measurement method are presented in detail. In Chapter 3, I present Super-resolution Optical Lithography with DNA (SOLiD), which is the super-resolution microscopy method that I developed to construct a nanoparticle assembly. In Chapter 4, the kinetics of single-stranded DNA folding and unfolding is discussed. Finally, in Chapter 5, the dissertation is concluded with future directions of this research.

Single-molecule Fluorescence Studies of Metalloregulators, Metalloenzymes, and Nanocatalysts

Author : Nesha May Octavio Andoy
Publisher :
ISBN 13 :
Total Pages : 274 pages
Book Rating : 4.:/5 (854 download)

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Book Synopsis Single-molecule Fluorescence Studies of Metalloregulators, Metalloenzymes, and Nanocatalysts by : Nesha May Octavio Andoy

Download or read book Single-molecule Fluorescence Studies of Metalloregulators, Metalloenzymes, and Nanocatalysts written by Nesha May Octavio Andoy and published by . This book was released on 2011 with total page 274 pages. Available in PDF, EPUB and Kindle. Book excerpt: This work presents the development and application of single-molecule fluorescence-based methodologies to study bio-inorganic systems and catalysis of nanostructured materials. Single-molecule fluorescence microscopy was used to examine: (i) protein-DNA interactions using single-molecule fluorescence resonance energy transfer (smFRET), (ii) single-molecule enzymology of tyrosinase using fluorescence quenching via energy transfer, and (iii) structure-activity correlation of gold nanoparticle catalysis using super-resolution imaging. Engineered DNA Holliday junction (HJ) was used as the smFRET reporter for interactions of a metalloregulator, CueR, with DNA for transcriptional regulation. Both the apo- and holo-forms of CueR interact with and change the structures and dynamics of interconversion between the two conformations of the HJ. Singlemolecule kinetic analyses revealed differences between apo- and holo- CueR in their interactions with DNA. These changes observed on HJ upon CueR binding reflect the protein actions on DNA for regulating gene transcription. The single-molecule enzymology of tyrosinase was also investigated. Catalysis coupled fluorescence quenching (CCFQ) was used to monitor the state of the binuclear-copper active site of the enzyme. The intermediates of this metal center exhibit different absorption features as the enzyme goes through the catalytic cycle. Using a fluorescent probe whose fluorescence spectrum overlaps with the absorption spectrum of one of the intermediates of the active site, the state of the active site of the enzyme was directly monitored. The structure-activity correlation of single nanoparticle catalysts was achieved by applying super-resolution fluorescence imaging on single-particle catalysis. The generation of a fluorescent product from a non-fluorescent reactant on the surface of nanoparticles was imaged using single-molecule fluorescence microscopy. Precise nanometer localization of where the products are generated, with a resolution of ~25 nm, was obtained by fitting the point spread function of the emission of a product molecule with a two-dimensional Gaussian function. By taking the SEM image of the same nanoparticles and overlaying them to those obtained from super-resolution imaging, direct correlation of the activity and structure of nanocatalysts was achieved at the single-particle level.

Polarized Super-resolution Fluorescence Microscopy

Author : César Augusto Valadés Cruz
Publisher :
ISBN 13 :
Total Pages : 253 pages
Book Rating : 4.:/5 (913 download)

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Book Synopsis Polarized Super-resolution Fluorescence Microscopy by : César Augusto Valadés Cruz

Download or read book Polarized Super-resolution Fluorescence Microscopy written by César Augusto Valadés Cruz and published by . This book was released on 2014 with total page 253 pages. Available in PDF, EPUB and Kindle. Book excerpt: While super-resolution microscopy has brought a significant improvement in nanoscale imaging of molecular assemblies in biological media, its extension to imaging molecular orientation using fluorescence anisotropy has not yet been fully explored. Providing orientational order information at the nanoscale would be of considerable interest for the understanding of biological functions since they are intrinsically related to structural fundamental processes such as in protein clustering in cell membranes, supra-molecular polymerization or aggregation. In this thesis, we propose a super-resolution polarization-resolved microscopy technique able to image molecular orientation behaviors in static and dynamic environments, in order to report structural information at the single molecule level and at nanometric spatial scale. Using direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with polarized detection, fluorescence anisotropy images are reconstructed at a spatial resolution of a few tens of nanometers. We analyze numerically the principle of the method in combination with models for orientational order mechanisms, and provide conditions for which this information can be retrieved with high precision in biological samples based on fibrillar structures. Finally, we propose an alternative technique based on stochastic fluctuations of single molecules: polarized super-resolution optical fluctuation imaging (polar-SOFI), and compare this approach with the previous one. We illustrate both techniques on molecular order imaging in actin stress fibers and tubulin fibers in fixed cells, DNA fibers and insulin amyloid fibrils.

Fluorescence in Bio-inspired Nanotechnology

Author : Jonas Hannestad
Publisher : Springer Science & Business Media
ISBN 13 : 3319010689
Total Pages : 127 pages
Book Rating : 4.3/5 (19 download)

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Book Synopsis Fluorescence in Bio-inspired Nanotechnology by : Jonas Hannestad

Download or read book Fluorescence in Bio-inspired Nanotechnology written by Jonas Hannestad and published by Springer Science & Business Media. This book was released on 2013-07-20 with total page 127 pages. Available in PDF, EPUB and Kindle. Book excerpt: In his thesis Fluorescence in Bio-inspired Nanotechnology, Jonas Hannestad describes the evolving field of DNA nanotechnology in a lucid and easily accessible way. A central theme in the thesis is how biological structures and mechanisms constitute a basis for the design of novel technologies. Hannestad discusses how self-assembled, nanometer-scale DNA constructs can be functionalized using fluorescent labeling. In particular, he highlights how applications are based on fluorescence resonance energy transfer (FRET). Another important contribution is the development of a lipid monolayer platform for the step-by-step assembly of DNA nanoconstructs. The work in the thesis is based on five peer-reviewed papers published in high-profile journals, all of which involve major contributions from the author.

Structural Organization and Chemical Activity Revealed by New Developments in Single-molecule Fluorescence and Orientation Imaging

Download Structural Organization and Chemical Activity Revealed by New Developments in Single-molecule Fluorescence and Orientation Imaging PDF Online Free

Author : Tianben Ding
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (137 download)

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Book Synopsis Structural Organization and Chemical Activity Revealed by New Developments in Single-molecule Fluorescence and Orientation Imaging by : Tianben Ding

Download or read book Structural Organization and Chemical Activity Revealed by New Developments in Single-molecule Fluorescence and Orientation Imaging written by Tianben Ding and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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 of single fluorescent molecules. I first present an SM approach for resolving spatial variations in the catalytic activity of individual photocatalysts. Quantitative colocalization of chemically triggered molecular probes reveals the role of structural defects on the activity of catalytic nanoparticles. Next, I demonstrate a new engineered optical point spread function (PSF), called the Duo-spot PSF, for SM orientation measurements. This PSF exhibits high sensitivity for estimating orientations of dim fluorescent molecules. This dissertation also discusses a new amyloid imaging method, transient amyloid binding (TAB) microscopy, for studying heterogeneous organization of amyloid structures, which are associated with various aging-related neurodegenerative diseases. Continuous transient binding of dye molecules to amyloid structures generates photon bursts for SM localization over hours to days with minimal photobleaching, yielding about 40% more localizations than standard immunolabeling. Finally, I augment TAB imaging to simultaneously measure positions and orientations of fluorescent molecules bound to amyloid surfaces. This new method, termed single-molecule orientation localization microscopy (SMOLM), robustly and sensitively measures the in-plane (xy) orientations of fluorophores (approximately 9 degree precision in azimuthal angle) near a refractive index interface and reveals structural heterogeneities along amyloid fibrillar networks that cannot be resolved by SM localization alone.

Single Molecule Sensing Beyond Fluorescence

Author : Warwick Bowen
Publisher : Springer Nature
ISBN 13 : 3030903397
Total Pages : 426 pages
Book Rating : 4.0/5 (39 download)

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Book Synopsis Single Molecule Sensing Beyond Fluorescence by : Warwick Bowen

Download or read book Single Molecule Sensing Beyond Fluorescence written by Warwick Bowen and published by Springer Nature. This book was released on 2022-03-01 with total page 426 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides an interesting snapshot of recent advances in the field of single molecule nanosensing. The ability to sense single molecules, and to precisely monitor and control their motion is crucial to build a microscopic understanding of key processes in nature, from protein folding to chemical reactions. Recently a range of new techniques have been developed that allow single molecule sensing and control without the use of fluorescent labels. This volume provides an overview of recent advances that take advantage of micro- and nanoscale sensing technologies and provide the prospect for rapid future progress. The book endeavors to provide basic introductions to key techniques, recent research highlights, and an outlook on big challenges in the field and where it will go in future. It is a valuable contribution to the field of single molecule nanosensing and it will be of great interest to graduates and researchers working in this topic.

Single-molecule Fluorescence Microscopy Studies of DNA-surface Interactions on Chemically Graded Organosilane Surfaces

Author : Zi Li
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (18 download)

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Book Synopsis Single-molecule Fluorescence Microscopy Studies of DNA-surface Interactions on Chemically Graded Organosilane Surfaces by : Zi Li

Download or read book Single-molecule Fluorescence Microscopy Studies of DNA-surface Interactions on Chemically Graded Organosilane Surfaces written by Zi Li and published by . This book was released on 2018 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation describes the application of wide-field single-molecule fluorescence microscopy techniques to investigations of DNA-surface interactions on chemically graded organosilane surfaces. The adsorption and desorption behaviors of double-stranded (ds) plasmid DNA along the amino-trimethoxysilane and octyl-trichlorosilane gradients were explored as a function of solution pH, solution ionic strength and surface properties. The results provide an improved fundamental understanding of DNA interactions with different surfaces and are certain to aid in the development and advancement of DNA-based biological and biomedical devices. Three distinct experiments were performed in completion of the work for this dissertation. In the first study, total internal reflection fluorescence (TIRF) microscopy was employed to study DNA interactions with aminosilane gradient surfaces under relatively acidic and basic environments. Electrical potentials were applied to assist DNA adsorption and desorption. The single-molecule data clearly showed that DNA capture and release was achieved on the monolayer and submonolayer coated regions of the aminosilane gradient surface under relatively basic pH conditions, with the help of an electrical potential. Meanwhile, DNA adsorption was found to be quasi-reversible on the multilayers at the high aminosilane end of the gradient in the relatively acidic solution. The results of these studies demonstrate the importance of manipulating the electrostatic interactions of DNA with charged surfaces in order to achieve DNA capture and release. The fundamental knowledge of the DNA-surface interactions gained in these studies will be helpful in diverse fields ranging from the layer-by-layer assembly of polyelectrolyte-based thin films to the selective electronic sensing of charged biomolecules. In the second study, the local dielectric properties of the least polar environments in dsDNA were assessed by using the solvatochromic dye, nile red, as a polarity-sensitive probe. TIRF spectroscopic imaging methods were employed in these studies. Although the dielectric constant within the least polar regions of dsDNA was previously predicted by theoretical and computational methods, no experimental measurements of its value had been reported to date. The results provide important knowledge of the factors governing the polarity of DNA microenvironments to which intercalators bind, and provide vital experimental support for the values determined in computational studies. In the third study, TIRF microscopy and single molecule tracking methods were employed to study DNA interactions with an opposed two-component C8-silane and aminosilane gradient surface as a function of solution pH. The mobility of surface-adsorbed DNA molecules was explored and quantified in these studies. The preliminary results further demonstrated the importance of electrostatic interactions over hydrophobic interactions in governing the adsorption of DNA to surfaces. The mobility of surface-adsorbed DNA was found to be largely independent of position along the two-component gradient. These studies were originally undertaken as a route to observation of cooperative effects that are believed to govern DNA-surface binding. Unfortunately, no clear evidence of cooperative effects was observed at the mixed regions of the two-component gradient surface.