Basic Confocal Microscopy

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.

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.

There has been a great upsurge in interest in light microscopy in recent years due to the advent of a number of significant advances in microscopy, one of the most important of which is confocal microscopy. Confocal microscopy has now become an important research tool, with a large number of new fluorescent dyes becoming available in the past few years, for probing your pet structure or molecule within fixed or living cell or tissue sampies. Many of the people interested in using confocal microscopy to further their research do not have a background in microscopy or even cell biology and so not only do they find considerable difficulty in obtaining satisfactory results with a confocal microscope, but they may be mislead by how data is being presented. This book is intended to teach you the basic concepts ofmicroscopy, fluorescence, digital imaging and the principles of confocal microscopy so that you may take full advantage ofthe excellent confocal microscopes now available. This book is also an excellent reference source for information related to confocal microscopy for both beginners and the more advanced users. For example, do you need to know the optimal pinhole size for a 63x 1. 4 NA lens? Do you need to know the fluorescence emission spectrum of Alexa 568? Access to the wealth of practical information in this book is made easier by using both the detailed index and the extensive glossary.

This manual contains selected material from Cells - a Laboratory Manual, as well as two chapters from Live Cell Imaging. It includes sections on microscopy, and on preparing and labelling specimens for microscopy.

Confocal Microscopy: Methods and Protocols, Second Edition takes the researcher from the bench top through the imaging process, to the page. Protocols for the preparation of tissues from many model organisms including worms, flies and mice have been included as well as chapters on confocal imaging of living cells, three dimensional analysis, and the measurement and presentation of confocal images for publication. Emphasis has been placed on the laser scanning confocal microscope since this is still the instrument used for most routine applications. The current generation of modern confocal instruments produces optical sections of cells and tissues that are free of out-of-focus fluorescence with reduced chances of artifacts from the techniques of specimen preparation. This allows the imaging of living specimens and measurements of physiological events within cells. Confocal microscopy has become essential in many fields of contemporary biomedical research where a light microscope is required for imaging fluorescently labeled cells and tissues, especially cell biology, developmental biology, neurobiology, and pathology. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Confocal Microscopy: Methods and Protocols, Second Edition is aimed primarily, but not exclusively, at the novice user with pointers to more advanced techniques.

Describes the principles of the technique and illustrates applications in physical and biomedical sciences. Covers Image formation in confocal microscopy, Performance of the confocal microscope, Biological and industrial applications. Paper. DLC: Confocal microscopy.

This volume of the acclaimed Methods in Cell Biology series provides specific examples of applications of confocal microscopy to cell biological problems. It is an essential guide for students and scientists in cell biology, neuroscience, and many other areas of biological and biomedical research, as well as research directors and technical staff of microscopy and imaging facilities. An integrated and up-to-date coverage on the many various techniques and uses of the confocal microscope (CM). Includes detailed protocols accessible to new users Details how to set up and run a "Confocal Microscope Core Facility" Contains over 170 figures