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PREFACE

This is the second issue in the CRC Press book series entitled "Advances in Pathology, Microscopy, and Molecular Morphology." The book is dedicated to the presentation of the most promising and modern techniques of molecular morphology. It is a comprehensive guide and a practical laboratory protocols book; each technique is described and discussed by first-class authors, each of whom enjoys a worldwide reputation in the discipline of molecular morphology.

Molecular morphology is a field of microscopy, histology, and cytology that has emerged from the combination and adaptation of techniques formerly developed for immunology, biochemistry, and molecular biology. During the past decade, this field has grown enormously and evolved into a new discipline of scientific technologies and applications, useful for various areas of biomedical research and clinical diagnostics. Molecular morphology allows the microscopic visualization of biochemical/molecular biological/ physiologic processes in human, animal, and plant tissues, and the useful areas include diagnostic molecular pathology, histo- and cytogenetics, and manifold other fields of medicine and biology. From a purely analytical perspective, molecular and immunological techniques have now been adapted to in situ localization, thus allowing the precise and highly specific localization of substances such as peptides, proteins, DNA, and RNA, each readily visible via conventional or adapted light and electron microscopy.

The molecular pathologist today relies to a large degree on techniques of molecular morphology, thereby analyzing the location and possible alterations of substances, such as nucleic acid sequence amplification or deletion, detection of intrinsic or external genes, or immunological tumor markers demonstrated within cellular and subcellular structures. Use of these molecular tools makes the diagnosis and management of cancer, viral infections, and other diseases more precise and reliable. Furthermore, combining techniques of molecular morphology with the capabilities of modern computer imaging and processing has provided a third dimension now accessible in 3D microscopy. The latter application facilitates both the full-color demonstration of spatial structure and much more precise measurements within the images obtained in all three dimensions. The neurobiologist, for example, can apply ultrasensitive and specified amplified immunohistochemical methodologies to analyze (co)-localization and structural relationships of neuropeptide-containing terminal nerve fibers to a much more sensitive and more realistic degree than previously attainable. Applications and new technologies are being developed and modified continuously.

This book distinguishes itself from other books not only by the quality of contributors included, but also in the approach to the topic: it is a type of "cookbook" of latest technology. The most robust recent developments in the discipline have been assembled and presented in a very useful, practical way, not only for experienced researchers, but also for neophytes who want to carry out productive science by relying on state-of-the-art technology. Such techniques are usually presented singly in journals, and not often in reproducible format. Very often, authors "do not tell their secrets." In this book, they do, and they explain in detail firsthand how their techniques work and how experiments can be brought to a successful conclusion.

Each of the 18 chapters follows a special format, similar to the first book of this series, entitled Gold and Silver Staining: Techniques in Molecular Morphology. After an introduction and a complete and accurate listing of materials (with sources of each special reagent, Web sites, and other necessary data), detailed step-by-step protocols are given, followed by a description of how the results should look. A technical hints and discussion section, key references, and figures illustrating the appropriate outcome complete each chapter.

In Chapter 1, Guida Portela-Gomes, a highly reputed specialist for co-localization studies in neuroendocrinology, gives expert guidance for the practical use and application of double and multiple immunostaining techniques in peptide research and diagnosis. Chris van der Loos, the authority in multiple staining in biomedical research, provides in Chapter 2 a treasure source for everyone attempting successful combinations of various methodologies of immunohistochemistry. Recent advances in enzyme-based amplification methods, novel fluorophores, and improved detection protocols have dramatically increased the utility of fluorescence localization procedures in human tissue sections. Kevin Roth and Denis Baskin, both among the most respected authorities in histochemistry and cytochemistry, provide in Chapter 3 detailed protocols for enzyme-enhanced immunohistochemical and in situ hybridization detection and discuss practical solutions to common problems encountered using these sensitive methods. In Chapter 4, James Hainfeld and Richard Powell, the original inventors of nanogold particles and gold enhancement, provide a detailed overview of applications of clustered gold in molecular morphology. Chapter 5 is a technological highlight contributed by Raymond Tubbs and collaborators, describing a new versatile and reliable detection method for HER2 gene amplification facilitating the detection of certain cancer gene alterations in daily clinicalpathological diagnosis. Chapter 6 is dedicated to high-power signal amplification methods, by which single gene molecular detection sensitivity has been achieved. This text is contributed by the editors of this book, Gerhard Hacker and Raymond Tubbs. Microarray- based genomic hybridization as a tool for the survey of genomic alterations in human neoplasms is described in Chapter 7 by Dina Kandil and colleagues of the Cleveland Clinic. Shin-ichi Izumi and collaborators in Chapter 8 introduce their own technique, termed "southwestern histochemistry." The senior author of this chapter is Paul Nakane, the original inventor of the immunoperoxidase method and undoubtedly one of the world's top authorities in the field of molecular morphology.

Automated mRNA in situ hybridization and tissue microarrays are described in Chapter 9 by Hiro Nitta and colleagues. The methodology described here illustrates the utility of fully automated messenger RNA detection in large numbers of different tissues on a single tissue microarray slide, and therefore can be understood as an important contribution toward increased reproducibility, standardization, and economy. Chapter 10 concerns a new method of whole mount in situ hybridization, contributed by Sabine Tontsch and colleagues. In Chapter 11, Marek Skacel and collaborators open new doors to molecular cytopathology, outlining the methodology of interphase fluorescence in situ hybridization performed on liquid-based thin-layer cytopathologic preparations. Lisa Bobroski and Omar Bagasra in Chapter 12 overview the latest developments in in situ PCR and give detailed descriptions for successfully performing this technique. Omar Bagasra is the original inventor of in situ PCR.

Andreas Aschoff and Gustav Jirikowski in Chapter 13 describe their technique for the visualization of apoptotic markers in human biopsies in high resolution. In Chapter 14, Gerhard Hacker and co-authors introduce the 3D (three-dimensional) digital optical microscope technology. Numerous full-color 3D photomicrographs demonstrate applications of this method for molecular morphology and also give examples from general biology. Chapter 15, by Michelle Lennartz and colleagues, is dedicated to the visualization of signal transduction pathways in real time. Masahiko Zuka in Chapter 16 demonstrates his promising method to estimate the proteolytic activity by film in situ zymography. Chapter 17 is dedicated to a more ethically conscious production and purchase of reagents for molecular morphology. The medical biologist Gerhard Hacker, the biochemist Günter Schwamberger, and the attorney-at-law Antoine Goetschel in this chapter merge their three different special areas of expertise to articulate a humane viewpoint of reagent selection for molecular morphology. Last but not least, in Chapter 18, authored by Anthony Rhodes, this European pioneer of standardization and quality management in diagnostic immunocytochemistry reminds molecular morphologists of the preeminent importance of quality assurance when applying molecular techniques in diagnosis and research.

The contents of this book are intended to provide reproducible, practical information on the latest technology and offer a balanced view. From the beginning, we have emphasized the use of human tissues or cell-culture-derived preparations. With this, we would like to follow a direction already given in the first book of this series, emphasizing the avoidance of animal experiments whenever possible. Again, most contributors responded to our request to do so, and we would like to thank them for their enlightened contributions.

We would like to thank the distinguished authors of this book for sharing their experience with the readers. It is our intent that this publication serve as a valuable source of practical information in molecular morphology, and that it will be used in laboratories for many years to come.