Fundamentals Of Fluorescence Microscopy

Fundamentals of Fluorescence Microscopy

This book starts at an introductory level and leads reader to the most advanced topics in fluorescence imaging and super-resolution techniques that have enabled new developments such as nanobioimaging, multiphoton microscopy, nanometrology and nanosensors. The interdisciplinary subject of fluorescence microscopy and imaging requires complete knowledge of imaging optics and molecular physics. So, this book approaches the subject by introducing optical imaging concepts before going in more depth about advanced imaging systems and their applications. Additionally, molecular orbital theory is the important basis to present molecular physics and gain a complete understanding of light-matter interaction at the geometrical focus. The two disciplines have some overlap since light controls the molecular states of molecules and conversely, molecular states control the emitted light. These two mechanisms together determine essential imaging factors such as, molecular cross-section, Stoke shift, emission and absorption spectra, quantum yield, signal-to-noise ratio, Forster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP) and fluorescence lifetime. These factors form the basis of many fluorescence based devices. The book is organized into two parts. The first part deals with basics of imaging optics and its applications. The advanced part takes care of several imaging techniques and related instrumentation that are developed in the last decade pointing towards far-field diffraction unlimited imaging.

Fundamentals of Fluorescence Microscopy

Introduction to Fluorescence

The phenomenon known as ‘fluorescence’ is now widely used in the chemical, physical and life sciences largely due to the development of highly sophisticated fluorescent probe chemistries and the commercial availability of these probes as well as the development of novel microscopy approaches. This Second Edition of Introduction to Fluorescence helps readers acquire a thorough understanding of basic fluorescence theory and practice. It describes the general principles in a direct way and uses examples from a variety of disciplines to demonstrate them. Since the First Edition, significant advances in the field have appeared. For example, phasors, both lifetime and spectral phasors, have become very popular, and so a new chapter dedicated to this topic has been added in this edition. Furthermore, significant advances have been made in fluorescence microscopy methods, including super-resolution and single-molecule techniques. In color throughout, the book takes readers through the history of important discoveries to the most current advances. It introduces the fundamentals of the fluorescence phenomenon and gives detailed examples of fluorescence applications in the molecular life sciences, including biochemistry, biophysics, clinical chemistry and diagnostics, pharmaceutical science, and cell and molecular biology. The author presents the basic theories underlying the applications and offers in-depth information on practical aspects. Numerous references are given in each chapter, along with a list of particularly important references at the end of each chapter. The text incorporates more than 340 figures that clearly illustrate the concepts and gives the chemical structures of the most widely used fluorescent molecules. In addition, Chapter 13, the Appendix, provides a "Rogue’s Gallery" of the most common errors and pitfalls to avoid.