In the last decade, fluorescence microscopy has evolved from a classical “retrospective” microscopy approach into an advanced imaging technique that allows the observation of cellular activities in living cells with increased resolution and dimensions. A bright new future has arrived as the nano era has placed a whole new array of tools in the hands of biophysicists who are keen to go deeper into the intricacies of how biological systems work. Following an introduction to the complex world of optical microscopy, this book covers topics such as the concept of white confocal, nonlinear optical microscopy, fluctuation spectroscopies, site-specific labeling of proteins in living cells, imaging molecular physiology using nanosensors, measuring molecular dynamics, muscle braking and stem cell differentiation.

"Alberto Diaspro has been choreographing light's dance for over 20 years, and in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, he has assembled a diverse group of experts to explain the methods they use to coax light to reveal biology's secrets."- From the Foreword by Daniel Evanko, editor, Nature Methods Nanoscopy and Multidimens

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.

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.

Covers three main areas.The phenomenon of fluorescence, the main applications, and the most common problems.

Zu dem Thema gibt es viele Publikationen, die von Experten für Experten geschrieben wurden. Dieses Buch wendet sich insbesondere an Studenten höherer Semester und Forscher, denen das Hintergrundwissen der Physik fehlt, um neuartige Verfahren der Fluoreszenzmikroskopie zu verstehen. Die zweite Auflage wartet mit neuen Kapiteln und einer erweiterten Einführung auf. Der Schwerpunkt liegt auf der hochauflösenden und Einzelmolekül-Mikroskopie. Jedes Kapitel wurde von einem anerkannten Experten des Fachgebiets geschrieben und sorgfältig überarbeitet, um so die Entwicklungen der letzten Jahre wiederzugeben.

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