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Author: Camille Bayas Publisher: ISBN: Category : Languages : en Pages :
Book Description
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.
Author: Camille Bayas Publisher: ISBN: Category : Languages : en Pages :
Book Description
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.
Author: Alex von Diezmann Publisher: ISBN: Category : Languages : en Pages :
Book Description
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.
Author: Marissa Kim Lee Publisher: ISBN: Category : Languages : en Pages :
Book Description
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.
Author: Publisher: Elsevier ISBN: 0128164646 Category : Science Languages : en Pages : 404
Book Description
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
Book Description
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.
Author: Joshua Yoon Publisher: ISBN: Category : Languages : en Pages :
Book Description
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.
Author: Vasily Astratov Publisher: Springer Nature ISBN: 3030217221 Category : Science Languages : en Pages : 487
Book Description
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.
Author: Publisher: Academic Press ISBN: 0123814839 Category : Science Languages : en Pages : 745
Book Description
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
Author: Krishnarao Appasani Publisher: ISBN: 110853449X Category : Medical Languages : en Pages : 172
Book Description
A comprehensive volume that brings together authoritative overviews of single molecule science techniques from a biological perspective.
Author: Anda Cornea Publisher: Elsevier ISBN: 0124167136 Category : Science Languages : en Pages : 261
Book Description
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
Author: Camille Bayas
Author: Camille Bayas
Author: Alex von Diezmann
Author: Marissa Kim Lee
Author: 
Author: Clément Cabriel
Author: Joshua Yoon
Author: Vasily Astratov
Author: 
Author: Krishnarao Appasani
Author: Anda Cornea