Optical Fluorescence Microscopy

In the last decade, fluorescence microscopy has evolved from a classical “retrospective” microscopy approach into an advanced imaging technique that allows the observation of cellular activities in living cells with increased resolution and dimensions. A bright new future has arrived as the nano era has placed a whole new array of tools in the hands of biophysicists who are keen to go deeper into the intricacies of how biological systems work. Following an introduction to the complex world of optical microscopy, this book covers topics such as the concept of white confocal, nonlinear optical microscopy, fluctuation spectroscopies, site-specific labeling of proteins in living cells, imaging molecular physiology using nanosensors, measuring molecular dynamics, muscle braking and stem cell differentiation.

"Alberto Diaspro has been choreographing light’s dance for over 20 years, and in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, he has assembled a diverse group of experts to explain the methods they use to coax light to reveal biology’s secrets." — From the Foreword by Daniel Evanko, editor, Nature Methods Nanoscopy and Multidimensional Optical Fluorescence Microscopy demonstrates that the boundaries between sciences do blur at the bottom, especially those that might separate the optical work of physicists and the cellular work of microbiologists. In 18 chapters written by pioneering researchers, this work offers the first comprehensive and current documentation of the cutting-edge research being accomplished in a wide range of photonic devices with revolutionary application. The highlight of the book is its coverage of optical nanoscopy and super-resolution microscopy. The rapid advances in this area over the past few years offer researchers in both photonics and molecular biologya wealth of accomplishment upon which they can build. Offering a complete treatment of this emerging field, this volume: Describes how scientists have exploited the properties of light and its fluorophore partners to overcome the resolution limit of conventional light microscopy Delves into recent ways to minimize the photobleaching that has long hampered many methods including those that have the potential to capture previously unobtainable information on the movements of single molecules Discusses the principles, benefits, and implementation of fluorescence correlation spectroscopy and related methods, which simplifies analysis by limiting light to stationary focal points in a sample Considers the most basic as well as emerging methods for improving three-dimensional optical sectioning microscopy Reviews the basics of FRET (fluorescence resonance energy transfer) and considers its new use for investigating protein complexes The text also introduces those emerging nonfluorescence microscopy methods that can actually exert mechanical forces to trap and move a variety of objects ranging from beads to living cells and cellular organelles. Combining this technique with fluorescence microscopy provides an unparalleled ability to manipulate and visualize biological samples. In the half-century since Richard Feynman challenged scientists to come up with the tools to investigate and manipulate our world at the nanoscale, we have succeeded in placing tools in the hands of biophysicists that are leading to major breakthroughs in our understanding of life and our ability to diagnose, treat, and prevent many challenges to human health. This book reflects what has been accomplished to date while pointing the way to what still needs to be done.

This book provides a comprehensive account of the theory of image formation in a confocal fluorescence microscope as well as a practical guideline to the operation of the instrument, its limitations, and the interpretation of confocal microscopy data. The appendices provide a quick reference to optical theory, microscopy-related formulas and definitions, and Fourier theory.

In the last decade, fluorescence microscopy has evolved from a classical “retrospective” microscopy approach into an advanced imaging technique that allows the observation of cellular activities in living cells with increased resolution and dimensions. A bright new future has arrived as the nano era has placed a whole new array of tools in the hands of biophysicists who are keen to go deeper into the intricacies of how biological systems work. Following an introduction to the complex world of optical microscopy, this book covers topics such as the concept of white confocal, nonlinear optical microscopy, fluctuation spectroscopies, site-specific labeling of proteins in living cells, imaging molecular physiology using nanosensors, measuring molecular dynamics, muscle braking and stem cell differentiation.

While there are many publications on the topic written by experts for experts, this text is specifically designed to allow advanced students and researchers with no background in physics to comprehend novel fluorescence microscopy techniques. This second edition features new chapters and a subsequent focus on super-resolution and single-molecule microscopy as well as an expanded introduction. Each chapter is written by a renowned expert in the field, and has been thoroughly revised to reflect the developments in recent years.

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.

Presents a fully updated, self-contained textbook covering the core theory and practice of both classical and modern optical microscopy techniques.

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