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The story of microscopy over the years is one of wonder, revelation, and even love. What better words could there be to describe the amazing things that we have been able to see, learn and accomplish thanks to the progress made in this field? A love story between a pieace of glass and the rainbow with an original soundtrack mad of poetry and music. From Galilei’s initial foray into basic optical microscopy, including the Camillo Golgi and Giuliano Toraldo di Francia lessons, to such later developments as time-resolved microscopy, multi-photon microscopy and three-dimensional microscopy to innovations such as optical nanoscopy, bioimaging and super resolution imaging, the book seeks to take the reader, be they scientist or layperson, on a journey through the evolution of the microscope and its many uses, including in the field of medicine. The author uses visible light as a through-line to unite the various chapters, as well as using fluorescence as a touchpoint from which to map the changes in the science, a significant choice, as it, along with label-free approaches and the addition of artificial intelligence, form the natural environment for development of the modern multi-messenger microscope towards bioimaging at the nanoscale.

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.

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.

This monograph demonstrates the latest developments in two-photon fluorescence microscopy and second-harmonic generation (SHG) microscopy, including coverage of high-resolution microscopy methods, such as STED microscopy. A special focus lies on clinical applications of these methods, e.g. in dermatology, ophtalmology, neuro sciences and cell biology.