This new volume, number 123, of Methods in Cell Biology looks at methods for quantitative imaging in cell biology. It covers both theoretical and practical aspects of using optical fluorescence microscopy and image analysis techniques for quantitative applications. The introductory chapters cover fundamental concepts and techniques important for obtaining accurate and precise quantitative data from imaging systems. These chapters address how choice of microscope, fluorophores, and digital detector impact the quality of quantitative data, and include step-by-step protocols for capturing and analyzing quantitative images. Common quantitative applications, including co-localization, ratiometric imaging, and counting molecules, are covered in detail. Practical chapters cover topics critical to getting the most out of your imaging system, from microscope maintenance to creating standardized samples for measuring resolution. Later chapters cover recent advances in quantitative imaging techniques, including super-resolution and light sheet microscopy. With cutting-edge material, this comprehensive collection is intended to guide researchers for years to come. Covers sections on model systems and functional studies, imaging-based approaches and emerging studies Chapters are written by experts in the field Cutting-edge material
Cells perform a range of complex functions to maintain homeostasis, including regulation of gene expression, selective trafficking of molecules between subcellular compartments, and protein expression. These processes are mediated by dynamic complexes of proteins and other molecules. Quantitative imaging in biology is concerned with answering questions about the spatial distribution, dynamics and conformational changes of these complexes as they perform their biological functions. This study utilizes a range of quantitative imaging techniques--including plasmon rulers, quantitative fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and super-resolution imaging--to answer biologically relevant questions. Microorganisms often contend with fluctuating environmental conditions and shifting metabolic demands, and their survival depends on their ability to rapidly alter gene expression. In bacteria, rapid regulation of gene expression is facilitated by transcription attenuation and anti-termination mechanisms that involve the binding of proteins to RNA and the manipulation of RNA structure. In Bacillus species the trp RNA-binding Attenuation Protein (TRAP) modulates the expression of the tryptophan biosynthetic pathway by binding messenger RNA and interfering with transcription elongation. Chapter 2 describes work to characterize the mechanism of TRAP binding to RNA, utilizing a single-molecule method that employs RNA-linked pairs of gold nano-particles--plasmon rulers. Eukaryotic cells segregate their genetic material into an envelope-bound nucleus, and all transport and communication between this compartment and the cytoplasm is mediated by the nuclear pore complex (NPC), a large multi-protein channel. NPC-mediated transport of materials between the cytoplasm and the nucleus is essential for many basic cell functions. The components of this molecular machine have been characterized, and there are several unproven models that describe how these components might function in concert. However, the mechanism by which this system of molecules mediates selective, direction transport has yet to be elucidated. The nuclear transport receptor importin-[beta], as well as Ran and Nup153 have been shown to be necessary for modulating selectivity of active and passive transport through the NPC. This study provides mechanistic details about importin-[beta] interactions with the pore, which mediate selective, directional transport. Quantitative fluorescence microscopy, FRAP and super-resolution imaging are used to study the interplay of importin-[beta], Ran and Nup153 in regulating the selectivity and efficiency of the mammalian NPC. Chapter 3 describes the use of FRAP and inverse FRAP (iFRAP) to quantify the dynamics of importin-[beta] turnover in the nuclear pore complex. Chapter 4 describes the use of super-resolution microscopy to characterize the distribution of importin-[beta] in the NPC under a range of conditions. This study characterizes the thermodynamics and kinetics of importin-[beta] interaction with the NPC and shows how Ran and Nup153 mediate these interactions. Importin-[beta] is an integral part of the NPC gate, and Ran acts to remodel this gate. The nucleoporin Nup153 plays a critical in the mechanism, acting as a coordinating site for importin-[beta] and Ran action.
This book provides an essential overview of existing state-of-the-art quantitative imaging methodologies and protocols (intensity-based ratiometric and FLIM/ PLIM). A variety of applications are covered, including multi-parametric quantitative imaging in intestinal organoid culture, autofluorescence imaging in cancer and stem cell biology, Ca2+ imaging in neural ex vivo tissue models, as well as multi-parametric imaging of pH and viscosity in cancer biology. The current state-of-the-art of 3D tissue models and their compatibility with live cell imaging is also covered. This is an ideal book for specialists working in tissue engineering and designing novel biomaterial.
A diverse collection of state-of-the-art methods for the microscopic imaging of cells and molecules. The authors cover a wide spectrum of complimentary techniques, including such methods as fluorescence microscopy, electron microscopy, atomic force microscopy, and laser scanning cytometry. Additional readily reproducible protocols on confocal scanning laser microscopy, quantitative computer-assisted image analysis, laser-capture microdissection, microarray image scanning, near-field scanning optical microscopy, and reflection contrast microscopy round out this eclectic collection of cutting-edge imaging techniques now available. The authors also discuss preparative methods for particles and cells by transmission electron microscopy.
The previous edition of this book marked the shift in technology from video to digital camera use with microscope use in biological science. This new edition presents some of the optical fundamentals needed to provide a quality image to the digital camera. Specifically, it covers the fundamental geometric optics of finite- and infinity-corrected microscopes, develops the concepts of physical optics and Abbe’s theory of image formation, presents the principles of Kohler illumination, and finally reviews the fundamentals of fluorescence and fluorescence microscopy. The second group of chapters deals with digital and video fundamentals: how digital and video cameras work, how to coordinate cameras with microscopes, how to deal with digital data, the fundamentals of image processing, and low light level cameras. The third group of chapters address some specialized areas of microscopy that allow sophisticated measurements of events in living cells that are below the optical limits of resolution. Expands coverage to include discussion of confocal microscopy not found in the previous edition Includes "traps and pitfalls" as well as laboratory exercises to help illustrate methods
Quantitative bioimaging is a broad interdisciplinary field that exploits tools from biology, chemistry, optics, and statistical data analysis for the design and implementation of investigations of biological processes. Instead of adopting the traditional approach of focusing on just one of the component disciplines, this textbook provides a unique introduction to quantitative bioimaging that presents all of the disciplines in an integrated manner. The wide range of topics covered include basic concepts in molecular and cellular biology, relevant aspects of antibody technology, instrumentation and experimental design in fluorescence microscopy, introductory geometrical optics and diffraction theory, and parameter estimation and information theory for the analysis of stochastic data. Key Features: Comprises four parts, the first of which provides an overview of the topics that are developed from fundamental principles to more advanced levels in the other parts. Presents in the second part an in-depth introduction to the relevant background in molecular and cellular biology and in physical chemistry, which should be particularly useful for students without a formal background in these subjects. Provides in the third part a detailed treatment of microscopy techniques and optics, again starting from basic principles. Introduces in the fourth part modern statistical approaches to the determination of parameters of interest from microscopy data, in particular data generated by single molecule microscopy experiments. Uses two topics related to protein trafficking (transferrin trafficking and FcRn-mediated antibody trafficking) throughout the text to motivate and illustrate microscopy techniques. An online appendix providing the background and derivations for various mathematical results presented or used in the text is available at http://www.routledge.com/9781138598980.
This book offers a comprehensive selection of essays by leading experts, which covers all aspects of modern imaging, from its application and up-scaling to its development. The chapter content ranges from the basics to the most complex overview of method and protocols. There is ample practical and detailed "how-to" content on important, but rarely addressed topics. This first edition features all-colour-plate chapters, licensed software and a unique, continuously updated website forum.
Quantitative fluorescence microscopy is concerned with making measurements from fluorescent specimens in a fluorescence microscope, by measuring fluorescence emission from a defined area or areas of a specimen. This technique is most commonly used to determine the amount of some specific substance, such as DNA, in some particular area of a cell. But it has many other uses; for example, it can be used to identify certain substances in the cell by examining their fluorescence characteristics. This book is a complete guide to this technique for all biologists. It describes the principles and applications of quantitative fluorescence microscopy and also gives much practical information about the instrumentation required. There is also a discussion of the exciting developments in confocal fluorescence microscopy which allows the three dimensional distribution of particular substances to be determined. Everyone presently using this technique, or wishing to start using it will need to read this book.
