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 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.

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 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 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 High Speed Atomic Force Microscopy in Biology written by Toshio Ando and published by Springer Nature. This book was released on 2022-03-23 with total page 327 pages. Available in PDF, EPUB and Kindle. Book excerpt: This first book on high-speed atomic force microscopy (HS-AFM) is intended for students and biologists who want to use HS-AFM in their research. It provides straightforward explanations of the principle and techniques of AFM and HS-AFM. Numerous examples of HS-AFM studies on proteins demonstrate how to apply this new form of microscopy to specific biological problems. Several precautions for successful imaging and the preparation of cantilever tips and substrate surfaces will greatly benefit first-time users of HS-AFM. In turn, the instrumentation techniques detailed in Chapter 4 can be skipped, but will be useful for engineers and scientists who want to develop the next generation of high-speed scanning probe microscopes for biology. The book is intended to facilitate the first-time use of this new technique, and to inspire students and researchers to tackle their own specific biological problems by directly observing dynamic events occurring in the nanoscopic world. Microscopy in biology has recently entered a new era with the advent of high-speed atomic force microscopy (HS-AFM). Unlike optical microscopy, electron microscopy, and conventional slow AFM, it allows us to directly observe biological molecules in physiological environments. Molecular “movies” created using HS-AFM can directly reveal how molecules behave and operate, without the need for subsequent complex analyses and roundabout interpretations. It also allows us to directly monitor morphological change in live cells, and dynamic molecular events occurring on the surfaces of living bacteria and intracellular organelles. As HS-AFM instruments were recently commercialized, in the near future HS-AFM is expected to become a common tool in biology, and will enhance and accelerate our understanding of biological phenomena.

Download or read book Single molecule Localization Microscopy and Applications to Visualize the Accessible Genome with ATAC see written by Maurice Youzong Lee and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: It has been thirty years since the first experimental observation of a single molecule, and thirteen years since the first few single-molecule super-resolution fluorescence microscopy methods were first published. Since these two key milestones, scientists have been developing and applying new optical methods to better understand the world around us, whether it is in crystals at cryogenic temperatures or in the crowded environment within living cells. In single-molecule localization microscopy, we first use fluorescent molecules to label the biological structure we are interested in studying. Next, instead of imaging all the fluorescent molecules at the same time in a single camera frame, we can use chemical means to make the molecules stochastically blink on and off. In this manner, instead of separating the overlapping fluorescent spots (or point spread functions) from each single emitter in space, we can now separate them temporally. Next, we can perform a process called superlocalization to measure their precise molecular coordinates in each camera frame, before recombining them spatially to create a super-resolution reconstruction. In my dissertation, I will describe how I have developed new capabilities for single-molecule localization microscopy and how I combined our super-resolution imaging methods with a new accessible chromatin labeling scheme to try to image the accessible genome in the nucleus. First, I will describe my attempts in using an engineered point spread function (PSF) called the corkscrew PSF to image labeled structures in 3D, but with a faster acquisition rate with a higher density of emitters per camera frame and fewer overlapping PSFs as compared to other engineered PSFs. From my work, I found that we had to use real-time Z drift correction to ensure that the amount of spherical aberration in our optical systems do not change while imaging a sample. A varying amount of spherical aberration changes the behavior of the corkscrew PSF over the imaging period which makes it challenging to accurately and precisely measure the locations of the emitters. Second, I will describe how I have fabricated a transmissive fixed dielectric phase mask that encodes for the Tetrapod PSF for use in our 4f optical systems. Microfabrication is a process that has to be done properly to create a functioning end product. I have tried to describe it with as many details as possible in Chapter 4. The fixed phase masks that I fabricated encode for a Tetrapod PSF that has an effective working axial range of 6 μm and work for emission wavelengths of 660 nm or 550 nm, and they have been used in multiple projects in the lab that resulted in several publications. Finally, I will describe how I have used our super-resolution imaging methods to visualize the accessible genome in the nucleus that has been labeled with a biochemical labeling method known as Assay of Transposase-Accessible Chromatin with visualization (ATAC-see). The two-meter long human genome is heavily compacted into a ten-micron-wide nucleus. But a part of the genome has to remain accessible to the biomolecular machinery that carry out gene regulation, transcription, and DNA repair. It would be useful if we could image the accessible genome within the nucleus and compare this map of accessible chromatin with other maps of labeled biomolecules. ATAC-see labels accessible chromatin in the nucleus with fluorophores. We can image the labeled fluorescence distribution with single-molecule localization microscopy to obtain super-resolution images of the underlying structure of accessible chromatin in the nucleus. This project had many twists and turns but I did make several interesting observations that may help to improve the ATAC-see labeling method so that future scientists may be able to accurately and reproducibly image the accessible genome in the nucleus. One observation was that ATAC-see appears to preferentially label the periphery of the nucleus. Another observation is that the Tn5 monomers do not readily dimerize after binding to the fluorescently-labeled dsDNA sequencing adapters. This greatly reduces the concentration of effective labels by about 30 times. Two ways to promote dimerization may be to use a peptide linker to connect two Tn5 monomers together or to connect the dsDNA sequencing adapters via extending the "reverse" strand.

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 Single Molecule Tools Part A Fluorescence Based Approaches written by and published by Academic Press. This book was released on 2010-08-17 with total page 559 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 A: Fluorescence Based Approaches 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 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.

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 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 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 Encyclopedia of Cell Biology written by and published by Academic Press. This book was released on 2015-08-07 with total page 2972 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Encyclopedia of Cell Biology, Four Volume Set offers a broad overview of cell biology, offering reputable, foundational content for researchers and students across the biological and medical sciences. This important work includes 285 articles from domain experts covering every aspect of cell biology, with fully annotated figures, abundant illustrations, videos, and references for further reading. Each entry is built with a layered approach to the content, providing basic information for those new to the area and more detailed material for the more experienced researcher. With authored contributions by experts in the field, the Encyclopedia of Cell Biology provides a fully cross-referenced, one-stop resource for students, researchers, and teaching faculty across the biological and medical sciences. Fully annotated color images and videos for full comprehension of concepts, with layered content for readers from different levels of experience Includes information on cytokinesis, cell biology, cell mechanics, cytoskeleton dynamics, stem cells, prokaryotic cell biology, RNA biology, aging, cell growth, cell Injury, and more In-depth linking to Academic Press/Elsevier content and additional links to outside websites and resources for further reading A one-stop resource for students, researchers, and teaching faculty across the biological and medical sciences

Download or read book Fermentation Microbiology and Biotechnology Fourth Edition written by E. M. T. El-Mansi and published by CRC Press. This book was released on 2018-12-17 with total page 420 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fermentation Microbiology and Biotechnology, 4th Edition explores and illustrates the broad array of metabolic pathways employed for the production of primary and secondary metabolites, as well as biopharmaceuticals. This updated and expanded edition addresses the whole spectrum of fermentation biotechnology, from fermentation kinetics and dynamics to protein and co-factor engineering. It also sheds light on the new strategies employed by industrialist for increasing tolerance and endurance of microorganisms to the accumulation of toxic wastes in microbial-cell factories. The new edition builds upon the fine pedigree of its earlier predecessors and extends the spectrum of the book to reflect the multidisciplinary and buoyant nature of this subject area. Key Features Covers the whole spectrum of the field from fermentation kinetics to control of fermentation and protein engineering. Includes case studies specifically designed to illustrate industrial applications and current state-of-the-art technologies. Presents the contributions of eminent international academics and industrial experts. Offers new chapters addressing: The prospects and the role of bio-fuels refineries, Control of metabolic efflux to product formation in microbial-cell factories and Improving tolerance of microorganisms to toxic byproduct accumulation in the fermentation vessel.

Download or read book Fluorescence Spectroscopy and Microscopy in Biology written by Radek Šachl and published by Springer Nature. This book was released on 2023-04-27 with total page 532 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides the reader with an updated comprehensive view of the rapidly developing and fascinating field of fluorescence spectroscopy and microscopy. In recent years, fluorescence spectroscopy and microscopy have experienced rapid technological development, which has enabled the detection and monitoring of single molecules with high spatial and temporal resolution. Thanks to these developments, fluorescence has become an even more popular method in physical, biological and related fields. This book guides the reader through both basic and advanced fluorescence spectroscopy and microscopy approaches with a focus on their applications in membrane and protein biophysics. Each of the four parts: A - Fluorescence Spectroscopy, B - Fluorescence Microscopy, C - Applications of Fluorescence Spectroscopy and Microscopy to biological membranes and D - Applications of Fluorescence Spectroscopy to protein studies are written by experts within the field. The book is intended for both complete beginners who want to quickly orient themselves in the large number of existing fluorescent methods, as well as for advanced readers who are interested in particular methods and their proper use.

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.