Download or read book Three dimensional Super resolution Microscopy and Single particle Tracking of Bacterial Proteins written by Camille Bayas and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The first optical detection of a single molecule (SM) at cryogenic temperatures 30 years ago laid the groundwork for the routine detection of SMs today at biologically relevant temperatures, thus uncovering hidden heterogeneity that might be obscured by ensemble techniques. In addition to enabling studies of the intricate photochemistry and photophysics of fluorescent labels at the SM level, SM fluorescence has also proven useful for the imaging and tracking of cellular structures and biomolecules in a non-invasive manner with high sensitivity. The ability to genetically express fluorescent protein fusions in live cells has allowed specific labeling, and thus imaging and tracking, of dynamic processes and structures in cells. This dissertation describes applications of SM-based single-particle tracking (SPT) and super-resolution (SR) microscopy for the study of spatial organization and dynamics of bacterial proteins in two and three spatial dimensions. In an SPT experiment, the position of a SM emitter at very low concentration is measured over time to generate a trajectory, allowing for observation and quantification of labeled biomolecule dynamics at the SM level. In a SR microscopy experiment, the diffraction-limited (DL) resolution of a conventional fluorescence microscope (~200 nm in xy) is circumvented by temporally separating the emission of many SM emitters decorating a structure through control of their emissive state. A "super-resolved" image, with a factor of ~5-10 resolution improvement over a conventional DL fluorescence image, is generated by estimating the positions of many non-moving SM emitters over many frames and building up an image reconstruction in a pointillist manner. Chapter 1 of this dissertation provides an introduction to fluorescence, SM imaging, SM-based SR microscopy, and SPT. Chapter 1 also gives a brief introduction to Caulobacter crescentus, the bacterium used as the model organism in the SM studies in Chapters 4 and 5. Chapter 2 describes the experimental methods used to perform quantitative SM imaging of bacterial cells. The combination of SM imaging with point spread function (PSF) engineering has enabled the accurate and precise localization of SMs in three dimensions (3D) by the intentional introduction of specifically chosen aberrations in the emission path of an SM microscope. Throughout this dissertation, the double-helix (DH) PSF, a rotating PSF composed of two lobes whose angle encodes axial position, was used to estimate 3D SM positions. Chapter 2 describes the implementation of the DH-PSF via optical Fourier processing, and Chapter 3 describes the robust and comprehensible two-color Easy-DHPSF v2 software for localizing molecules in 3D and for registering localizations from two spectral channels into the same coordinate system with nanoscale accuracy. The resolution improvement gained from SM-based techniques is particularly useful for bacteria, the sizes of which are on the order of the DL. 3D SM-based SR and SPT have enabled the observation of structures and dynamics at length scales below the DL. Caulobacter is a useful biological target where understanding of the mechanisms for asymmetric cell division need to be explored and quantified. Central to Caulobacter's asymmetric division is the dynamic spatiotemporal regulation of gene expression and protein localization. Chapters 4 and 5 describes research performed in collaboration with Prof. Lucy Shapiro's laboratory (Department of Developmental Biology, Stanford School of Medicine) to study gene expression and signaling proteins in Caulobacter. Chapter 4 describes work studying the spatial organization and dynamics of ribosomes and a RNA-degrading enzyme RNase E using 3D SR microscopy and SPT. Results showed that the organization and dynamics of RNase E and ribosomes are closely related to the transcriptional activity of the cell. Finally, Chapter 5 describes SPT studies of the membrane-bound histidine kinase and stalked cell fate determinant DivJ in an effort to probe the physical properties of the Caulobacter stalked pole. Preliminary SPT results suggest that disrupting the physical properties and interactions at the stalked pole has an influence on DivJ diffusion and signaling.

Download or read book Three dimensional Single molecule Microscopy of Bacterial Regulatory Proteins Within a Pole localized Microdomain written by Alex von Diezmann and published by . This book was released on 2018 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Since the first optical detection of a single molecule 29 years ago, the development of single-molecule microscopy and spectroscopy has revolutionized the study of complex chemical systems. As reviewed in Chapter 1, by imaging and computationally localizing individual fluorescent dyes or proteins within a sample, their positions can be localized with typical precisions (10-40 nm) an order of magnitude or better than the optical diffraction limit of visible light (~250 nm laterally and ~500 nm axially). This technique is critical to super-resolution fluorescence microscopy and single-molecule tracking, which are now regularly used to measure the nanoscale structures, biomolecular motions, and stochastic chemical processes underlying the biology of cells. This dissertation comprises two intertwined single-molecule imaging projects: 1) optical and analytical methods development for three-dimensional (3D) single-molecule tracking and super-resolution microscopy, and 2) the application of these methods to understand the nanoscale organization and dynamics of proteins at the poles of the bacterium Caulobacter crescentus. Without modification, a single-molecule microscope only improves imaging resolution in the lateral (xy) dimension, but biological cells are intrinsically 3D. To improve the imaging resolution in z, the detection path of a standard widefield microscope can be modified using Fourier processing to encode z position in the pattern of light formed by each fluorescent emitter and detected on the camera. Chapter 2 reviews the development of a two-color 3D single-molecule microscope that uses the double-helix point spread function pattern to encode 3D position, while Chapter 3 describes how to correctly align and to calibrate the fine aberrations of such a microscope to achieve nanoscale imaging accuracy in multiple color channels simultaneously. The bacterium Caulobacter crescentus is a model organism for the study of cell polarization and asymmetric cell division. Chapters 4 and 5 describe work performed in collaboration with Prof. Lucy Shapiro and her laboratory in the Department of Developmental Biology in the Stanford University School of Medicine to study how the tips, or poles, of Caulobacter cells use proteins to act as nanoscale spatial landmarks that polarize cells and induce spatially organized development. The polar organizing protein PopZ is one such critical landmark, and Chapter 4 describes results obtained from 3D super-resolution imaging of PopZ. Such imaging showed that PopZ forms 150-200 nm space-filling polar microdomains of roughly uniform density, and that proteins of the chromosome partitioning machinery (ParA and ParB) exhibit different spatial behaviors (recruitment vs. tethering) relative to the PopZ microdomain depending on their biochemistry and role in the chromosome replication process. Chapter 5 discusses the combination of single-molecule tracking and super-resolution imaging to study the polar localization of the signaling molecules of that activate the master regulator protein CtrA. Precise 3D imaging and tracking showed that PopZ acts as a selectively permeable localization hub that slows the motion of signaling proteins. In combination with reaction-diffusion modeling and transcriptional assays, these microscopic measurements indicated that the PopZ microdomain acts to sequester the CtrA signaling pathway within the pole and spatially pattern transcriptional output within the predivisional Caulobacter cell.

Download or read book Single molecule and Super resolution Microscopy of Bacterial Cells written by Marissa Kim Lee and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Single molecules were first detected at low temperatures twenty-six years ago in the laboratory of W.E. Moerner. Subsequent technological advances have allowed researchers to study single molecules at room temperatures and within living cells, providing novel biological insight about underlying spatial and dynamical heterogeneity. By combining single molecule detection with the ability to control the emissive state of the fluorescent label (also called "active control"), a suite of super-resolution imaging techniques has been developed. These single-molecule-based super-resolution imaging strategies leverage the fluorescence microscope's ability to non-invasively study multiple targets within living cells, while bridging the resolution gap between optical and electron microscopies. In large part, future advances to improve single molecule and super-resolution imaging require better fluorophore and labeling technologies. Utilizing fluorophore with higher photon yields will increase the resolution of super-resolution images and the data acquisition speed. Additionally, a greater library fluorophores with different of colors and sensing capabilities will enable application to more imaging targets. Currently, many single molecule and super-resolution experiments within living systems use fluorescent proteins because the labeling of target proteins is more straightforward. However, the limited photon yield of fluorescent proteins often results in tantalizingly fuzzy super-resolution images. Imaging the same targets, labeled instead with brighter organic emitters, could provide more image detail, but better fluorogenic and genetically encoded labeling schemes must be developed and discovered. The first chapter of this dissertation will introduce and discuss the historical context and basic principles of single molecule and super-resolution imaging. Chapter 2 will then describe the general experimental procedures necessary for quantitative single molecule and super-resolution imaging, including quantifying the number of photons detected (and emitted) from a single molecule, as well as the preparation of bacterial samples for fluorescence microscopy. Later chapters apply these fundamental experimental measurements to study bacterial biology and fluorophore photophysics. Chapters 3 and 4 concern the development and characterization of organic emitters suitable for single molecule or super-resolution imaging, work achieved with the synthetic collaboration of organic chemists in the laboratory of Professor Robert J. Twieg at Kent State University. Chapter 3 discusses the optimization of rhodamine spirolactam photoswitching such that activation could occur at visible wavelengths. The optimized rhodamine spirolactams were then covalently attached to the surface of bacterial cells and imaged with three-dimensional super-resolution. Images of the bacterial cell surface demonstrates a marked improvement in labeling uniformity, specificity, and density compared to previous methods which labeled the surface with the transient binding of a membrane sensitive dye. Chapter 4 introduces a novel enzyme-based strategy to control the fluorescence from nitro-aryl fluorogens. A proof-of-principle experiment demonstrated that endogenous nitroreductase enzymes within bacterial cells could catalyze the fluorescence-activating reaction, thus generating free fluorophores, which were detectable on the single-molecule-level within the cell. Lastly, chapter 5 summarizes three-dimensional imaging experiments (performed in collaboration with the laboratory of Professor Lucy Shapiro in the Department of Developmental Biology at Stanford University) of components of the bacterial gene expression machinery labeled with fluorescent proteins. Super-resolution imaging is ideally suited to the small size scale of bacterial cells, and a wealth of biological insights remains to be discovered. Simultaneously improving fluorophore photon yield, specificity, and active control strategies will have a profound impact on super-resolution precision and speed.

Download or read book Spectroscopy and Dynamics of Single Molecules written by and published by Elsevier. This book was released on 2019-08-14 with total page 404 pages. Available in PDF, EPUB and Kindle. Book excerpt: Spectroscopy and Dynamics of Single Molecules: Methods and Applications reviews the most recent developments in spectroscopic methods and applications. Spectroscopic techniques are the chief experimental methods for testing theoretical models and research in this area plays an important role in stimulating new theoretical developments in physical chemistry. This book provides an authoritative insight into the latest advances in the field, highlighting new techniques, current applications, and potential future developments An ideal reference for chemists and physicists alike, Spectroscopy and Dynamics of Single Molecules: Methods and Applications is a useful guide for all those working in the research, design, or application of spectroscopic tools and techniques across a wide range of fields. - Includes the latest research on ultrafast vibrational and electronic dynamics, nonlinear spectroscopies, and single-molecule methods - Makes the content accessible to researchers in chemistry, biophysics, and chemical physics through a rigorous multi-disciplinary approach - Provides content edited by a world-renowned chemist with more than 30 years of experience in research and instruction

Download or read book Three dimensional and Multicolour Approaches in Super resolution Fluorescence Microscopy for Biology written by Clément Cabriel and published by . This book was released on 2019 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell biology relies on imaging tools to provide structural and dynamic information about samples. Among them, fluorescence microscopy offers a compromise between high specificity and low toxicity. Recently, super-resolution methods overcame the diffraction barrier to unlock new fields of investigation. Single molecule approaches prove especially useful for three-dimensional nanoscale imaging, and allow couplings between different detection modalities. Still, their use is hindered by the complexity of the methods as well as the lack of reproducibility between experiments.We propose new methods to render super-localisation microscopy more easily applicable to relevant studies in cell biology, chemistry and material science. First, we introduce dedicated protocols and samples to eliminate sources of error in calibration and performance measurement acquisitions. We also provide examples of uses of three-dimensional super-localisation for state-of-the-art studies in the frameworks of cell adhesion and bacterial resistance to drugs.Then, we focus on the development of a novel optical method that provides unbiased results in three-dimensional single molecule localisation microscopy. This is achieved through the combination of two complementary axial detection strategies: point spread function shaping on the one hand, and supercritical angle fluorescence detection on the other hand. By cross-correlating and merging the lateral and axial positions provided by the different sources, we achieve quasi-isotropic localisation precisions down to 15 nanometres over a 1-micrometre capture range. We demonstrate the insensibility of the method to imaging non-idealities such as axial drift, chromatic aberration and sample tilt, and we propose applications in neurobiology and bacteria labelling.Finally, we introduce two new post-processing approaches for the demixing of simultaneous multi-species acquisitions. They are based respectively on the measurement of the spot sizes, and on the assessment of the dynamic blinking behaviour of molecules. After demonstrating a proof of principle, we assess the impact of the different parameters likely to influence the results. Eventually, we discuss leads to improve the demixing performances, and we discuss the coupling possibilities with complementary single molecule localisation techniques.

Download or read book Observing Structures and Dynamic Behavior in Biological Cells Using Single molecule Based Super resolution Fluorescence Microscopy written by Joshua Yoon and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: For the last three decades, the ability to detect single molecules at high spatiotemporal resolutions has revolutionized the way we observe and understand the cells that harbor life. This research uses super-resolution imaging and single-molecule tracking to uncover nanoscale structural details and dynamics for mammalian cells and bacteria. By optically separating out each individual emitter in time using an active-control mechanism, every localization provides spatial information with a resolution much better than the diffraction limit to yield super-resolution microscopy. To address the fact that biological systems are inherently three-dimensional, the microscope detection path is further extended to include a "4f system" configuration, which provides easy access to the conjugate back focal plane. By strategically placing a phase mask here, the emission can be optically transformed in way which breaks the symmetry of the detected intensity profile of a single-molecule emitter above and below the focal plane to give precise axial positions. However, it still remains a challenge to obtain a clear picture of the surface features of small, crowded biological structures in their natural habitat in both a non-invasive and precise manner. This dissertation describes how super-resolution fluorescence microscopy and surface meshing algorithms are used in conjunction to quantify the surface topology of two main biological systems: the primary cilium of mammalian cells and the surface of the bacterium, Caulobacter crescentus.

Download or read book Label Free Super Resolution Microscopy written by Vasily Astratov and published by Springer Nature. This book was released on 2019-08-31 with total page 487 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents the advances in super-resolution microscopy in physics and biomedical optics for nanoscale imaging. In the last decade, super-resolved fluorescence imaging has opened new horizons in improving the resolution of optical microscopes far beyond the classical diffraction limit, leading to the Nobel Prize in Chemistry in 2014. This book represents the first comprehensive review of a different type of super-resolved microscopy, which does not rely on using fluorescent markers. Such label-free super-resolution microscopy enables potentially even broader applications in life sciences and nanoscale imaging, but is much more challenging and it is based on different physical concepts and approaches. A unique feature of this book is that it combines insights into mechanisms of label-free super-resolution with a vast range of applications from fast imaging of living cells to inorganic nanostructures. This book can be used by researchers in biological and medical physics. Due to its logically organizational structure, it can be also used as a teaching tool in graduate and upper-division undergraduate-level courses devoted to super-resolved microscopy, nanoscale imaging, microscopy instrumentation, and biomedical imaging.

Download or read book Single Molecule Tools Part B Super Resolution Particle Tracking Multiparameter and Force Based Methods written by and published by Academic Press. This book was released on 2010-07-09 with total page 745 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and "just look at the thing" as anticipated by the visionary Richard Feynman already half a century ago. Single Molecule Tools, Part B: Super-Resolution, Particle Tracking, Multiparameter, and Force Based Methods captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools. - Includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools - Methods included are useful to both established researchers and newcomers to the field - Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines

Download or read book Single Molecule Science written by Krishnarao Appasani and published by . This book was released on 2022-05-11 with total page 172 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.

Download or read book Fluorescence Microscopy written by Anda Cornea and published by Elsevier. This book was released on 2014-02-24 with total page 261 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fluorescence Microscopy: Super-Resolution and other Novel Techniques delivers a comprehensive review of current advances in fluorescence microscopy methods as applied to biological and biomedical science. With contributions selected for clarity, utility, and reproducibility, the work provides practical tools for investigating these ground-breaking developments. Emphasizing super-resolution techniques, light sheet microscopy, sample preparation, new labels, and analysis techniques, this work keeps pace with the innovative technical advances that are increasingly vital to biological and biomedical researchers. With its extensive graphics, inter-method comparisons, and tricks and approaches not revealed in primary publications, Fluorescence Microscopy encourages readers to both understand these methods, and to adapt them to other systems. It also offers instruction on the best visualization to derive quantitative information about cell biological structure and function, delivering crucial guidance on best practices in related laboratory research. - Presents a timely and comprehensive review of novel techniques in fluorescence imaging as applied to biological and biomedical research - Offers insight into common challenges in implementing techniques, as well as effective solutions

Download or read book Microscope Image Processing written by Fatima Merchant and published by Academic Press. This book was released on 2022-08-26 with total page 528 pages. Available in PDF, EPUB and Kindle. Book excerpt: Microscope Image Processing, Second Edition, introduces the basic fundamentals of image formation in microscopy including the importance of image digitization and display, which are key to quality visualization. Image processing and analysis are discussed in detail to provide readers with the tools necessary to improve the visual quality of images, and to extract quantitative information. Basic techniques such as image enhancement, filtering, segmentation, object measurement, and pattern recognition cover concepts integral to image processing. In addition, chapters on specific modern microscopy techniques such as fluorescence imaging, multispectral imaging, three-dimensional imaging and time-lapse imaging, introduce these key areas with emphasis on the differences among the various techniques.The new edition discusses recent developments in microscopy such as light sheet microscopy, digital microscopy, whole slide imaging, and the use of deep learning techniques for image segmentation and analysis with big data image informatics and management.Microscope Image Processing, Second Edition, is suitable for engineers, scientists, clinicians, post-graduate fellows and graduate students working in bioengineering, biomedical engineering, biology, medicine, chemistry, pharmacology and related fields, who use microscopes in their work and would like to understand the methodologies and capabilities of the latest digital image processing techniques or desire to develop their own image processing algorithms and software for specific applications. - Presents a unique practical perspective of state-of-the-art microscope image processing and the development of specialized algorithms - Each chapter includes in-depth analysis of methods coupled with the results of specific real-world experiments - Co-edited by Kenneth R. Castleman, world-renowned pioneer in digital image processing and author of two seminal textbooks on the subject

Download or read book Far Field Optical Nanoscopy written by Philip Tinnefeld and published by Springer. This book was released on 2015-02-07 with total page 340 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book describes developments in the field of super-resolution fluorescence microscopy or nanoscopy. In 11 chapters, distinguished scientists and leaders in their respective fields describe different nanoscopy approaches, various labeling technologies, and concrete applications. The topics covered include the principles and applications of the most popular nanoscopy techniques STED and (f)PALM/STORM, along with advances brought about by fluorescent proteins and organic dyes optimized for fluorescence nanoscopy. Furthermore, the photophysics of fluorescent labels is addressed, specifically for improving their photoswitching capabilities. Important applications are also discussed, such as the tracking and counting of molecules to determine acting forces in cells, and quantitative cellular imaging, respectively, as well as the mapping of chemical reaction centers at the nano-scale. The 2014 Chemistry Nobel Prize® was awarded for the ground-breaking developments of super-resolved fluorescence microscopy. In this book, which was co-edited by one of the prize winners, readers will find the most recent developments in this field.

Download or read book Single Molecule Image Analysis written by Christian Franke and published by Frontiers Media SA. This book was released on 2022-12-15 with total page 152 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Super Resolution Microscopy written by Udo J. Birk and published by John Wiley & Sons. This book was released on 2017-09-12 with total page 466 pages. Available in PDF, EPUB and Kindle. Book excerpt: This unique book on super-resolution microscopy techniques presents comparative, in-depth analyses of the strengths and weaknesses of the individual approaches. It was written for non-experts who need to understand the principles of super-resolution or who wish to use recently commercialized instruments as well as for professionals who plan to realize novel microscopic devices. Explaining the practical requirements in terms of hardware, software and sample preparation, the book offers a wealth of hands-on tips and practical tricks to get a setup running, provides invaluable help and support for successful data acquisition and specific advice in the context of data analysis and visualization. Furthermore, it addresses a wide array of transdisciplinary fields of applications. The author begins by outlining the joint efforts that have led to achieving super-resolution microscopy combining advances in single-molecule photo-physics, fluorophore design and fluorescent labeling, instrument design and software development. The following chapters depict and compare current main standard techniques such as structured illumination microscopy, single-molecule localization, stimulated emission depletion microscopy and multi-scale imaging including light-sheet and expansion microscopy. For each individual approach the experimental setups are introduced, the imaging protocols are provided and the various applications illustrated. The book concludes with a discussion of future challenges addressing issues of routine applications and further commercialization of the available methods. Guiding users in how to make choices for the design of their own experiments from scratch to promising application, this one-stop resource is intended for researchers in the applied sciences, from chemistry to biology and medicine to physics and engineering.

Download or read book Single Molecule Spectroscopy written by R. Rigler and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 375 pages. Available in PDF, EPUB and Kindle. Book excerpt: The topics range from single molecule experiments in quantum optics and solid-state physics to analogous investigations in physical chemistry and biophysics.

Download or read book Single Biomolecule Detection and Analysis written by Tuhin Subhra Santra and published by CRC Press. This book was released on 2023-08-15 with total page 355 pages. Available in PDF, EPUB and Kindle. Book excerpt: This collection discusses various micro/nanodevice design and fabrication for single-biomolecules detection. It will be an ideal reference text for graduate students and professionals in diverse subject areas including materials science, biomedical engineering, chemical engineering, mechanical engineering, and nanoscience. This book- Discusses techniques of single-biomolecule detection, their advantages, limitations, and applications. Covers comprehensively several electrochemical detection techniques. Provides single-molecule separation, sensing, imaging, sequencing, and analysis in detail. Examines different types of cantilever-based biomolecule sensing, and its limitations. Single Biomolecule Detection and Analysis covers single-biomolecule detection and characterization using micro/nanotechnologies and micro/nanofluidic devices, electrical and magnetic detection technologies, microscopy and spectroscopy techniques, single biomolecule optical, and nanopore devices. The text covers key important biosensors-based detection, stochastic optical reconstruction microscopy-based detection, electrochemical detection, metabolic engineering of animal cells, single-molecule intracellular delivery and tracking, terahertz spectroscopy-based detection, total internal reflection fluorescence (TIFR) detection, and Fluorescence Correlation Spectroscopy (FCS) detection. The text will be useful for graduate students and professionals in diverse subject areas including materials science, biomedical engineering, chemical engineering, mechanical engineering, and nanoscience. Discussing chemical process, physical process, separation, sensing, imaging, sequencing, and analysis of single-molecule detection, this text will be useful for graduate students and professionals in diverse subject areas including materials science, biomedical engineering, chemical engineering, mechanical engineering, and nanoscience. It covers microscopy and spectroscopy techniques for single-biomolecule detection, analysis, and their biomedical engineering applications.

Download or read book Liquid Cell Electron Microscopy written by Frances M. Ross and published by Cambridge University Press. This book was released on 2017 with total page 529 pages. Available in PDF, EPUB and Kindle. Book excerpt: 2.6.2 Electrodes for Electrochemistry