This book encompasses the full breadth of the super-resolution imaging field, representing modern techniques that exceed the traditional diffraction limit, thereby opening up new applications in biomedicine. It shows readers how to use the new tools to increase resolution in sub-nanometer-scale images of living cells and tissue, which leads to new information about molecules, pathways and dynamics. The book highlights the advantages and disadvantages of the techniques, and gives state-of-the-art examples of applications using microscopes currently available on the market. It covers key techniques such as stimulated emission depletion (STED), structured illumination microscopy (SSIM), photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). It will be a useful reference for biomedical researchers who want to work with super-resolution imaging, learn the proper technique for their application, and simultaneously obtain a solid footing in other techniques.

This book encompasses the full breadth of the super-resolution imaging field, representing modern techniques that exceed the traditional diffraction limit, thereby opening up new applications in biomedicine. It shows readers how to use the new tools to increase resolution in sub-nanometer-scale images of living cells and tissue, which leads to new information about molecules, pathways and dynamics. The book highlights the advantages and disadvantages of the techniques, and gives state-of-the-art examples of applications using microscopes currently available on the market. It covers key techniques such as stimulated emission depletion (STED), structured illumination microscopy (SSIM), photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). It will be a useful reference for biomedical researchers who want to work with super-resolution imaging, learn the proper technique for their application, and simultaneously obtain a solid footing in other techniques.

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. Content Level Research

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 Multidimensional Optical Fluorescence Microscopy demonstrates that the boundaries between sciences do blur at the bottom, especially those that might separate the optical work of physicists and the cellular work of microbiologists. In 18 chapters written by pioneering researchers, this work offers the first comprehensive and current documentation of the cutting-edge research being accomplished in a wide range of photonic devices with revolutionary application. The highlight of the book is its coverage of optical nanoscopy and super-resolution microscopy. The rapid advances in this area over the past few years offer researchers in both photonics and molecular biologya wealth of accomplishment upon which they can build. Offering a complete treatment of this emerging field, this volume: * Describes how scientists have exploited the properties of light and its fluorophore partners to overcome the resolution limit of conventional light microscopy * Delves into recent ways to minimize the photobleaching that has long hampered many methods including those that have the potential to capture previously unobtainable information on the movements of single molecules * Discusses the principles, benefits, and implementation of fluorescence correlation spectroscopy and related methods, which simplifies analysis by limiting light to stationary focal points in a sample * Considers the most basic as well as emerging methods for improving three-dimensional optical sectioning microscopy * Reviews the basics of FRET (fluorescence resonance energy transfer) and considers its new use for investigating protein comple

"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 Multidimensional Optical Fluorescence Microscopy demonstrates that the boundaries between sciences do blur at the bottom, especially those that might separate the optical work of physicists and the cellular work of microbiologists. In 18 chapters written by pioneering researchers, this work offers the first comprehensive and current documentation of the cutting-edge research being accomplished in a wide range of photonic devices with revolutionary application. The highlight of the book is its coverage of optical nanoscopy and super-resolution microscopy. The rapid advances in this area over the past few years offer researchers in both photonics and molecular biologya wealth of accomplishment upon which they can build. Offering a complete treatment of this emerging field, this volume: Describes how scientists have exploited the properties of light and its fluorophore partners to overcome the resolution limit of conventional light microscopy Delves into recent ways to minimize the photobleaching that has long hampered many methods including those that have the potential to capture previously unobtainable information on the movements of single molecules Discusses the principles, benefits, and implementation of fluorescence correlation spectroscopy and related methods, which simplifies analysis by limiting light to stationary focal points in a sample Considers the most basic as well as emerging methods for improving three-dimensional optical sectioning microscopy Reviews the basics of FRET (

1. A Curious premise "My grandma was a beautiful woman...". This chapter tells about the motivation to decide to do research in life and why with the optical microscope. 2. Just observe! The optical microscope to observe living systems, from organs to proteins. The challenge from its invention to "tomorrow" to decipher cancer and neurological disorders. 3.The colours of the rainbow We all live under the rainbow, colours are delivering the energy needed to explore the living by watching. 4. The sharpener of the light When a curved piece of glass meets the light allows to see those fine dietails hidden to the eyes. 5. A three-dimensional world Flatlandia is a novel, the real world is developed along three spatial dimensions and the optical microscope can produce three-dimensional animated "postcard" by simply changing the lens focus when observing around. 6. Modern times: the space and time of observations Time is the fourth dimension that increases the budget of information at our disposal to understand what's going on at different time time and space scales. 7. Two photon are better than one Quantum mechanics allows to start a joyful revolution in optical microscopy with relevant implicantions in medicine and biology. Two photon is a unique entity. 8. Super eyes to see beyond physical limits Laws of physics limit the perfomances of the light microscope. No doubts. The image reconstrution channel has no limits if you are able to add information and the optical microscope an unlimited super power to visualize details. 9. Without a net Now is time to remove the net. We are skilled enough. So lets control the shape of light to get information without fluorescent labes. 10. The liquid microscope of the future Illumination produces multiple messages tuning across time and space scales and artificial intelligence can merge them to deciphering nature. Liquid tunable microscopy could provide the opportunity to see things differently and to change our point of view, abandoning the obsession of representing the "real world" we have in mind when forming an image. Lets see further! 11. Pop microscopy "Grown-ups never understand anything on their own, and it is tiring for children to always have to give them explanations. "We use nice images to bring you to instruments and applications like in a pop song that people whistle in the shower. 12. Acknowledgments It is a love narration in the love story between a curved piece of glass and the rainbow.

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.