This text guides you through the principles and practical techniques of confocal and multiphoton microscopy. It also describes the historical connections and parallel inventions that resulted in modern techniques of live cell imaging and their use in biology and medicine. You will find comparisons of different types of confocal and multiphoton microscopes, solutions to the problems one would encounter when using various microscopic techniques, tips on selecting equipment, and an extensive annotated bibliography of additional resources.

In the last decade multiphoton excitation microscopy has emerged as an important technique with ever increasing numbers of significant applications in the fields of biology, chemistry, physics, and medicine. This volume contains key papers on the following topics: developments of nonlinear optical spectroscopy and nonlinear scanning microscopy (SHG, CARS); theory and techniques of multiphoton excitation microscopy; development of laser sources; single-molecule studies; and applications to biology, cell biology, embryology and developmental biology, neuroscience, dermatology, and optical biopsy. A comprehensive bibliography follows the reprinted papers.

Fluorescence spectroscopy continues its advance to more sophisticated methods and applications. As one looks over the previous decades, its appears that the first practical instruments for time-resolved measurements appeared in the 1970’s. The instrumentation and analysis methods for time-resolved fluorescence advanced rapidly throughout the 1980’s. Since 1990 we have witnessed a rapid migration of the principles of time-resolved fluorescence to cell biology and clinical appli- tions. Most recently, we have seen the introduction of multi-photon excitation, pump-probe and stimulated emission methods for studies of biological mac- molecules and for cellular imaging. These advanced topics are the subject of the present volume. Two-photon excitation was first predicted by Maria Goppert-Mayer in 1931, but was not experimentally observed until 1961. Observation of two-photon excitation required the introduction of lasers which provided adequate photon density for multi-photon absorption. Since the early observations of two-photon excitation in the 1960s, multi-photon spectroscopy has been limited to somewhat exotic applications of chemical physics, where it is used to study the electronic symmetry of small molecules. Placing one’s self back in 1980, it would be hard to imagine the use of multi-photon excitation in biophysics or cellular imaging.

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

Advances in technology have revolutionized the development of light microscopy techniques in biomedical research, thus improving visualization of the microstructure of cells and tissues under physiological conditions. Fluorescence microscopy methods are non-contact and non-invasive and provide high spatial and temporal resolution that other laboratory techniques cannot. This well-illustrated book targets graduate students and scientists who are new to the state-of-the-art fluorescence microscopy techniques used in biological and clinical imaging. It explains basic concepts and imaging procedures for wide-field, confocal, multiphoton excitation, fluorescence resonance energy transfer (FRET), lifetime imaging (FLIM), spectral imaging, fluorescence recovery after photobleaching (FRAP), optical tweezers, total internal reflection, high spatial resolution atomic force microscopy (AFM), and bioluminescence imaging for gene expression. The usage of these techniques in various biological applications, including calcium, pH, membrane potential, mitochondrial signaling, protein-protein interactions under various physiological conditions, and deep tissue imaging, is clearly presented. The authors describe the approaches to selecting epifluorescence microscopy, the detectors, and the image acquisition and processing software for different biological applications. Step-by-step directions on preparing different digital formats for light microscopy images on websites are also provided.

This guide provides extensive coverage of microscopic imaging principles. After reviewing the main principles of image formation, diffraction, interference, and polarization used in microscopy, this guide describes the most widely applied microscope configurations and applications. It also covers major system components, including light sources, illumination layouts, microscope optics, and image detection electronics. This guide also provides a comprehensive overview of microscopy techniques, including bright field and dark field imaging, contrast enhancement methods (such as phase and amplitude contrast), DIC, polarization, and fluorescence microscopy. In addition, it describes scanning techniques (such as confocal and multiphoton imaging points); new trends in super-resolution methods (such as 4Pi microscopy, STED, STORM, and structured illumination); and array microscopy, CARS, and SPIM.

This open access book provides a comprehensive overview of the application of the newest laser and microscope/ophthalmoscope technology in the field of high resolution imaging in microscopy and ophthalmology. Starting by describing High-Resolution 3D Light Microscopy with STED and RESOLFT, the book goes on to cover retinal and anterior segment imaging and image-guided treatment and also discusses the development of adaptive optics in vision science and ophthalmology. Using an interdisciplinary approach, the reader will learn about the latest developments and most up to date technology in the field and how these translate to a medical setting. High Resolution Imaging in Microscopy and Ophthalmology – New Frontiers in Biomedical Optics has been written by leading experts in the field and offers insights on engineering, biology, and medicine, thus being a valuable addition for scientists, engineers, and clinicians with technical and medical interest who would like to understand the equipment, the applications and the medical/biological background. Lastly, this book is dedicated to the memory of Dr. Gerhard Zinser, co-founder of Heidelberg Engineering GmbH, a scientist, a husband, a brother, a colleague, and a friend.

This third edition of a classic text in biological microscopy includes detailed descriptions and in-depth comparisons of parts of the microscope itself, digital aspects of data acquisition and properties of fluorescent dyes, the techniques of 3D specimen preparation and the fundamental limitations, and practical complexities of quantitative confocal fluorescence imaging. Coverage includes practical multiphoton, photodamage and phototoxicity, 3D FRET, 3D microscopy correlated with micro-MNR, CARS, second and third harmonic signals, ion imaging in 3D, scanning RAMAN, plant specimens, practical 3D microscopy and correlated optical tomography.

The Handbook of Biomedical Nonlinear Optical Microscopy provides comprehensive treatment of the theories, techniques, and biomedical applications of nonlinear optics and microscopy for cell biologists, life scientists, biomedical engineers, and clinicians. The chapters are separated into basic and advanced sections, and provide both textual and graphical illustrations of all key concepts. The more basic sections are aimed at life scientists without advanced training in physics and mathematics, and tutorials are provided for the more challenging sections. The first part of the Handbook introduces the historical context of nonlinear microscopy. The second part presents the nonlinear optical theory of two- and multiphoton excited fluorescence (TPE, MPE) spectroscopy, second and third harmonic generation (SHG, THG) spectroscopy, and coherent anti-Stokes Raman spectroscopy (CARS). The third part introduces modern microscopic and spectroscopic instrumentation and techniques that are based on nonlinear optics. The fourth part provides key applications of nonlinear microscopy to the biomedical area: neurobiology, immunology, tumor biology, developmental biology, dermatology, and cellular metabolism. There are also chapters on nonlinear molecular probes, cellular damage, and nanoprocessing.

These are exciting times for the field of optical imaging of brain function. Rapid developments in theory and technology continue to considerably advance understanding of brain function. Reflecting changes in the field during the past five years, the second edition of In Vivo Optical Imaging of Brain Function describes state-of-the-art techniques and their applications for the growing field of functional imaging in the live brain using optical imaging techniques. New in the Second Edition: Voltage-sensitive dyes imaging in awake behaving animals Imaging based on genetically encoded probes Imaging of mitochondrial auto-fluorescence as a tool for cortical mapping Using pH-sensitive dyes for functional mapping Modulated imaging Calcium imaging of neuronal activity using 2-photon microscopy Fourier approach to optical imaging Fully updated chapters from the first edition Leading Authorities Explore the Latest Techniques Updated to reflect continuous development in this emerging research area, this new edition, as with the original, reaches across disciplines to review a variety of non-invasive optical techniques used to study activity in the living brain. Leading authorities from such diverse areas as biophysics, neuroscience, and cognitive science present a host of perspectives that range from a single neuron to large assemblies of millions of neurons, captured at various temporal and spatial resolutions. Introducing techniques that were not available just a few years ago, the authors describe the theory, setup, analytical methods, and examples that highlight the advantages of each particular method.