Introduction To Confocal Fluorescence Microscopy

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.

Basic Confocal Microscopy, Second Edition builds on the successful first edition by keeping the same format and reflecting relevant changes and recent developments in this still-burgeoning field. This format is based on the Confocal Microscopy Workshop that has been taught by several of the authors for nearly 20 years and remains a popular workshop for gaining basic skills in confocal microscopy. While much of the information concerning fluorescence and confocal microscopy that made the first edition a success has not changed in the six years since the book was first published, confocal imaging is an evolving field and recent advances in detector technology, operating software, tissue preparation and clearing, image analysis, and more have been updated to reflect this. Several of these advances are now considered routine in many laboratories, and others such as super resolution techniques built on confocal technology are becoming widely available.

This book discusses the various principles in confocal scanning microscopy which has become a useful tool in many practical fields including biological studies and industrial inspection. The methodology presented in this book is unique and is based on the concept of the three-dimensional transfer functions which have been developed by the author and his colleagues over the last five years. With the 3-D transfer functions, resolving power in 3-D confocal imaging can be defined in a unified way, different optical arrangements can be compared with an insight into their inter-relationship, and images of thick objects can be modeled in terms of the Fourier transform which makes the analysis easy. The aim of this book is to provide a systematic introduction to the concept of the 3-D transfer functions in various confocal microscopes, to describe the methods for the derivation of different 3-D transfer functions, and to explain the principles of 3-D confocal imaging in terms of these functions.

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.

Most researchers agree that biological confocal microscopy was jump-started by the confocal design first published by White and Amos in 1985 in the Journal of Cell Biology. As a result, this remains a relatively young field. Yet the use of the technique has grown phenomenally since those early efforts, with new users joining the ranks daily. The publication of Basic Confocal Microscopy reflects the burgeoning need to train new students, technologists, and faculty wishing to use confocal microscopy in their research. A direct outgrowth of the authors’ five-day intensive course in the subject begun in 2005, this book covers the basics and includes all the information required to design, implement, and interpret the results of, biological experiments based on confocal microscopy. Concise yet comprehensive, the volume begins by covering the core issues of fluorescence, specimen preparation and labeling, before moving on to address the analog-to-digital conversion of specimen data gathered using confocal microscopy. Subsequent chapters detail the practicalities of operating confocal microscopes, providing all the information necessary to begin practicing confocal microscopy as well as optimizing the material obtained. The final block of chapters examine 3-dimensional analysis and the reconstruction of data sets, outline some of the ethical considerations in confocal imaging, and then supply a number of resources that the authors have found useful in their own work. Once readers have mastered the information this book presents, the resources found in its pages will be an excellent guide to continued learning about the more advanced forms of confocal microscopy.

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

Confocal and Two-Photon Microscopy Foundations, Applications, and Advances Edited by Alberto Diaspro Confocal and two-photon fluorescence microscopy has provided researchers with unique possibilities of three-dimensional imaging of biological cells and tissues and of other structures such as semiconductor integrated circuits. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances provides clear, comprehensive coverage of basic foundations, modern applications, and groundbreaking new research developments made in this important area of microscopy. Opening with a foreword by G. J. Brakenhoff, this reference gathers the work of an international group of renowned experts in chapters that are logically divided into balanced sections covering theory, techniques, applications, and advances, featuring: * In-depth discussion of applications for biology, medicine, physics, engineering, and chemistry, including industrial applications * Guidance on new and emerging imaging technology, developmental trends, and fluorescent molecules * Uniform organization and review-style presentation of chapters, with an introduction, historical overview, methodology, practical tips, applications, future directions, chapter summary, and bibliographical references * Companion FTP site with full-color photographs * The significant experience of pioneers, leaders, and emerging scientists in the field of confocal and two-photon excitation microscopy Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances is invaluable to researchers in the biological sciences, tissue and cellular engineering, biophysics, bioengineering, physics of matter, and medicine, who use these techniques or are involved in developing new commercial instruments.