Fret And Flim Techniques

This volume reviews the techniques Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) providing researchers with step by step protocols and handy hints and tips. Both have become staple techniques in many biological and biophysical fields.

Introduces readers to the enlightening world of the modern light microscope There have been rapid advances in science and technology over the last decade, and the light microscope, together with the information that it gives about the image, has changed too. Yet the fundamental principles of setting up and using a microscope rests upon unchanging physical principles that have been understood for years. This informative, practical, full-colour guide fills the gap between specialised edited texts on detailed research topics, and introductory books, which concentrate on an optical approach to the light microscope. It also provides comprehensive coverage of confocal microscopy, which has revolutionised light microscopy over the last few decades. Written to help the reader understand, set up, and use the often very expensive and complex modern research light microscope properly, Understanding Light Microscopy keeps mathematical formulae to a minimum—containing and explaining them within boxes in the text. Chapters provide in-depth coverage of basic microscope optics and design; ergonomics; illumination; diffraction and image formation; reflected-light, polarised-light, and fluorescence microscopy; deconvolution; TIRF microscopy; FRAP & FRET; super-resolution techniques; biological and materials specimen preparation; and more. Gives a didactic introduction to the light microscope Encourages readers to use advanced fluorescence and confocal microscopes within a research institute or core microscopy facility Features full-colour illustrations and workable practical protocols Understanding Light Microscopy is intended for any scientist who wishes to understand and use a modern light microscope. It is also ideal as supporting material for a formal taught course, or for individual students to learn the key aspects of light microscopy through their own study.

The detection and measurement of the dynamic interactions of proteins within the living cell are critical to our understanding of cell physiology and pathophysiology. With FRET microscopy and spectroscopy techniques, basic and clinical scientists can make such measurements at very high spatial and temporal resolution. But sources of background information about these tools are very limited, so this book fills an important gap. It covers both the basic concepts and theory behind the various FRET microscopy and spectroscopy techniques, and the practical aspects of using the techniques and analyzing the results. The critical tricks for obtaining a good FRET image and precisely quantitating the signals from living specimens at the nanomolecular level are explained. Valuable information about the preparation of biological samples used for FRET image analysis is also provided. The methods covered include different types of microscopy systems and detectors (wide-field, confocal, multi-photon) as well as specialized techniques such as photobleaching FRET, FLIM-FRET microscopy, spectral imaging FRET, single molecule FRET, and time and image correlation spectroscopy. Starting from the basics, the chapters guide readers through the choice of probes to be used for FRET experiments and the selection of the most suitable experimental approaches to address specific biological questions. Up-to-date, consistently organized, and well-illustrated, this unique book will be welcomed by all researchers who wish to use FRET microscopy and spectroscopy techniques.

With contributions by numerous experts

Time-correlated Single Photon Counting has been written in the hope that by relating the authors' experiences with a variety of different single photon counting systems, they may provide a useful service to users and potential users of this formidably sensitive technique. Of all the techniques available to obtain information on the rates of depopulation of excited electronic singlet states of molecular species, monitoring of fluorescence provides, in principle, the simplest and most direct measure of concentration. This volume comprises eight chapters, with the first focusing on the time dependence and applications of fluorescence. Succeeding chapters go on to discuss basic principles of the single photon counting lifetime measurement; light sources; photomultipliers; electronics; data analysis; nanosecond time-resolved emission spectroscopy; time dependence of fluorescence anisotropy. This book will be of interest to practitioners in the field of chemistry.

This monograph focuses on modern femtosecond laser microscopes for two photon imaging and nanoprocessing, on laser tweezers for cell micromanipulation as well as on fluorescence lifetime imaging (FLIM) in Life Sciences. The book starts with an introduction by Dr. Wolfgang Kaiser, pioneer of nonlinear optics and ends with the chapter on clinical multiphoton tomography, the novel high resolution imaging technique. It includes a foreword by the nonlinear microscopy expert Dr. Colin Sheppard. Contents Part I: Basics Brief history of fluorescence lifetime imaging The long journey to the laser and its use for nonlinear optics Advanced TCSPC-FLIM techniques Ultrafast lasers in biophotonics Part II: Modern nonlinear microscopy of live cells STED microscopy: exploring fluorescence lifetime gradients for super-resolution at reduced illumination intensities Principles and applications of temporal-focusing wide-field two-photon microscopy FLIM-FRET microscopy TCSPC FLIM and PLIM for metabolic imaging and oxygen sensing Laser tweezers are sources of two-photon effects Metabolic shifts in cell proliferation and differentiation Femtosecond laser nanoprocessing Cryomultiphoton imaging Part III: Nonlinear tissue imaging Multiphoton Tomography (MPT) Clinical multimodal CARS imaging In vivo multiphoton microscopy of human skin Two-photon microscopy and fluorescence lifetime imaging of the cornea Multiscale correlative imaging of the brain Revealing interaction of dyes and nanomaterials by multiphoton imaging Multiphoton FLIM in cosmetic clinical research Multiphoton microscopy and fluorescence lifetime imaging for resection guidance in malignant glioma surgery Non-invasive single-photon and multi-photon imaging of stem cells and cancer cells in mouse models Bedside assessment of multiphoton tomography

Advances in technology have revolutionized the development of light microscopy techniques in biomedical research, thus improving visualization of the microstructure of cells and tissues under physiological conditions. Fluorescence microscopy methods are non-contact and non-invasive and provide high spatial and temporal resolution that other laboratory techniques cannot. This well-illustrated book targets graduate students and scientists who are new to the state-of-the-art fluorescence microscopy techniques used in biological and clinical imaging. It explains basic concepts and imaging procedures for wide-field, confocal, multiphoton excitation, fluorescence resonance energy transfer (FRET), lifetime imaging (FLIM), spectral imaging, fluorescence recovery after photobleaching (FRAP), optical tweezers, total internal reflection, high spatial resolution atomic force microscopy (AFM), and bioluminescence imaging for gene expression. The usage of these techniques in various biological applications, including calcium, pH, membrane potential, mitochondrial signaling, protein-protein interactions under various physiological conditions, and deep tissue imaging, is clearly presented. The authors describe the approaches to selecting epifluorescence microscopy, the detectors, and the image acquisition and processing software for different biological applications. Step-by-step directions on preparing different digital formats for light microscopy images on websites are also provided.

Interdisciplinarity is more often invoked than practised. This is hardly surprising, considering the daunting vastness of modern biology. To reach a satisfactory understanding of a complex biological system, a wide spectrum of conceptual and experimental tools must be applied at different levels, from the molecular to the cellular, tissue and organismic. We believe the multifaceted regulatory interplay between integrin receptors and ion channels offers a rich and challenging field for researchers seeking broad biological perspectives. By mediating cell adhesion to the extracellular matrix, integrins regulate many developmental processes in the widest sense (from cell choice between differentiation and proliferation, to tissue remodeling and organogenesis). Rapidly growing evidence shows that frequent communication takes place between cell adhesion receptors and channel proteins. This may occur through formation of multiprotein membrane complexes that regulate ion fluxes as well as a variety of intracellular signaling pathways. In other cases, cross talk is more indirect and mediated by cellular messengers such as G proteins. These interactions are reciprocal, in that ion channel stimulation often controls integrin activation or expression. From a functional standpoint, studying the interplay between integrin receptors and ion channels clarifies how the extracellular matrix regulates processes as disparate as muscle excitability, synaptic plasticity and lymphocyte activation, just to mention a few. The derangement of these processes has many implications for pathogenesis processes, in particular for tumor invasiveness and some cardiovascular and neurologic diseases. This book provides a general introduction to the problems and methods of this blossoming field.