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 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 Techniques written by Paul R. Selvin and published by CSHL Press. This book was released on 2008 with total page 511 pages. Available in PDF, EPUB and Kindle. Book excerpt: Geared towards research scientists in structural and molecular biology, biochemistry, and biophysics, this manual will be useful to all who are interested in observing, manipulating and elucidating the molecular mechanisms and discrete properties of macromolecules.

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 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 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 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 Handbook of Single Molecule Biophysics written by Peter Hinterdorfer and published by Springer Science & Business Media. This book was released on 2009-12-24 with total page 634 pages. Available in PDF, EPUB and Kindle. Book excerpt: This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.

Download or read book Bacterial Chromatin written by Remus T. Dame and published by Springer Nature. This book was released on with total page 654 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Molecular Biology of the Cell written by and published by . This book was released on 2002 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Single Molecule Optical Detection Imaging and Spectroscopy written by W. E. Moerner and published by John Wiley & Sons. This book was released on 2008-09-26 with total page 264 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single Molecule Spectroscopy is one of the hottest topics in today's chemistry. It brings us close to the the most exciting vision generations of chemists have been dreaming of: To observe and examine single molecules! While most of chemistry deals with myriads of molecules, this books presents the latest developments for the detection and investigation of single entities. Written by internationally renowned authors, it is a thorough and comprehensive survey of current methods and their applications.

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.

Download or read book Live Cell Imaging written by Robert D. Goldman and published by CSHL Press. This book was released on 2005 with total page 658 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent advances in imaging technology reveal, in real time and great detail, critical changes in living cells and organisms. This manual is a compendium of emerging techniques, organized into two parts: specific methods such as fluorescent labeling, and delivery and detection of labeled molecules in cells; and experimental approaches ranging from the detection of single molecules to the study of dynamic processes in organelles, organs, and whole animals. Although presented primarily as a laboratory manual, the book includes introductory and background material and could be used as a textbook in advanced courses. It also includes a DVD containing movies of living cells in action, created by investigators using the imaging techniques discussed in the book. The editors, David Spector and Robert Goldman, whose previous book was Cells: A Laboratory Manual,are highly respected investigators who have taught microscopy courses at Cold Spring Harbor Laboratory, the Marine Biology Laboratory at Woods Hole, and Northwestern University.

Download or read book Single Molecule Spectroscopy in Chemistry Physics and Biology written by Astrid Graslund and published by Springer Science & Business Media. This book was released on 2010-04-17 with total page 570 pages. Available in PDF, EPUB and Kindle. Book excerpt: Written by the leading experts in the field, this book describes the development and current state of the art in single molecule spectroscopy. The application of this technique, which started 1989, in physics, chemistry and biosciences is displayed.

Download or read book Engineering New Capabilities Into Optical Microscopes written by Matthew D. Lew and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The labeling of specific biological structures with single fluorescent molecules has ushered in a new era of imaging technology: super-resolution optical microscopy with resolution far beyond the diffraction limit down to some tens of nm. With the features of these exquisite tools in mind, this Dissertation discusses optical strategies for measuring the three-dimensional (3D) position and orientation of single molecules with nanoscale precision and several super-resolution imaging studies of structures in living cells. The concepts of single-molecule imaging, super-resolution microscopy, the engineering of optical point spread functions (PSFs), and quantitative analysis of single-molecule fluorescence images are introduced. The various computational methods and experimental apparatuses developed during the course of my graduate work are also discussed. Next, a new engineered point spread function, called the Corkscrew PSF, is shown for 3D imaging of point-like emitters. This PSF has been demonstrated to measure the location of nanoscale objects with 2-6 nm precision in 3D throughout a 3.2-micrometer depth range. Characterization and application of the Double-Helix (DH) PSF for super-resolution imaging of structures within mammalian and bacterial cells is discussed. The DH-PSF enables 3D single-molecule imaging within living cells with precisions of tens of nanometers throughout a ~2-micrometer depth range. Finally, the impact of single-molecule emission patterns and molecular orientation on optical imaging is treated, with particular emphasis on multiple strategies for improving the accuracy of super-resolution imaging. The DH microscope is shown to be well-suited for accurately and simultaneously measuring the 3D position and orientation of single molecules.

Download or read book Atomic Force Microscopy written by Ahmed Touhami and published by Morgan & Claypool Publishers. This book was released on 2020-05-11 with total page 113 pages. Available in PDF, EPUB and Kindle. Book excerpt: Over the last two decades, Atomic Force Microscopy (AFM) has undoubtedly had a considerable impact in unraveling the structures and dynamics of microbial surfaces with nanometer resolution, and under physiological conditions. Moreover, the continuous innovations in AFM-based modalities as well as the combination of AFM with modern optical techniques, such as confocal fluorescence microscopy or Raman spectroscopy, increased the diversity and volume of data that can be acquired in an experiment. It is evident that these combinations provide new ways to investigate a broad spectrum of microbiological processes at the level of single cells. In this book, I have endeavored to highlight the wealth of AFM-based modalities that have been implemented over the recent years leading to the multiparametric and multifunctional characterization of, specifically, bacterial surfaces. Examples include the real-time imaging of the nanoscale organization of cell walls, the quantification of subcellular chemical heterogeneities, the mapping and functional analysis of individual cell wall constituents, and the probing of the nanomechanical properties of living bacteria. It is expected that in the near future more AFM-based modalities and complementary techniques will be combined into single experiments to address pertinent problems and challenges in microbial research. Such improvements may make it possible to address the dynamic nature of many more microbial cell surfaces and their constituents, including the restructuring of cellular membranes, pores and transporters, signaling of cell membrane receptors, and formation of cell-adhesion complexes. Ultimately, manifold discoveries and engineering possibilities will materialize as multiparametric tools allow systems of increasing complexity to be probed and manipulated.

Download or read book Principles of Nano Optics written by Lukas Novotny and published by Cambridge University Press. This book was released on 2012-09-06 with total page 583 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fully revised and in its second edition, this standard reference on nano-optics is ideal for graduate students and researchers alike.