Flim Microscopy In Biology And Medicine

Detecting Signals at the Single Molecule Level: Pioneering Achievements in Microscopy Recent advances have led to such remarkable improvements in fluorescence lifetime imaging microscopy’s (FLIM) capacity for contrast and sensitivity that researchers can now employ it to detect signals at the single molecule level. FLIM also offers the additional benefit of independence from fluorophore concentration and excitation intensity. Moreover, its unique sensitivity makes it an excellent reporter of conformational changes and of variations in the molecular surroundings of biological molecules. Most of this improvement and discovery have occurred during the past decade, and, to date, information that would benefit a broad range of researchers remains scattered in the literature. Edited by two of the top pioneers in the field, FLIM Microscopy in Biology and Medicine presents the fundamentals of FLIM along with a number of advanced considerations so that a wider audience can appreciate recent and potential improvements that make it such a valuable tool. New Opportunities for Biomedical Researchers... New Challenges for Microscopy Researchers Discussion sections in all the chapters clearly show the challenges for implementing FLIM for various applications. Certain chapters discuss limits on the number of photons required for highly accurate lifetime determinations, as well as the accuracy with which multiple, closely associated lifetime components can reliably be determined. Such considerations are important for the user when he or she is selecting the most advantageous method of FLIM to use for a particular application. While this book provides an introduction for those new to FLIM, it gathers a wealth of material to enhance the work of experts involved in pioneering technological improvements, as well as those research opportunities in this unique and promising area of microscopy.

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.

Fluorescence microscopy images can be easily integrated into current video and computer image processing systems. People like visual observation; they like to watch a television or computer screen, and fluorescence techniques are thus becoming more and more popular. Since true in vivo experiments are simple to perform, samples can be directly seen and there is always the possibility of manipulating the samples during the experiments; it is an ideal technique for biology and medicine. Images are obtained by a classical (now called wide-field) fluorescence microscope, a confocal scanning microscope, upright or inverted, with epifluorescence or transmission. Computerized image processing may improve definition, and remove glare and scattered light signal. It also makes it possible to compute ratio images (ratio imaging both in excitation and in emission) or lifetime imaging. Image analysis programs may supply a great deal of additional data of various types, starting with calculations of the number of fluorescent objects, their shapes, brightness, etc. Fluorescence microscopy data may be complemented by classical measurement in the cuvette yr by flow cytometry.

This book is an attempt to bridge the gap between the instrumental principles of multi-dimensional time-correlated single photon counting (TCSPC) and typical applications of the technique. Written by an originator of the technique and by sucessful users, it covers the basic principles of the technique, its interaction with optical imaging methods and its application to a wide range of experimental tasks in life sciences and clinical research. The book is recommended for all users of time-resolved detection techniques in biology, bio-chemistry, spectroscopy of live systems, live cell microscopy, clinical imaging, spectroscopy of single molecules, and other applications that require the detection of low-level light signals at single-photon sensitivity and picosecond time resolution.

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.

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

The book reviews the recent developments in brain imaging and their technological advancements to understand molecular mechanisms associated with neurological disorders and basic behaviors in humans and rodents at the structural, molecular, and functional levels. It discusses the usefulness of advanced optical microscopy techniques, including optical coherence tomography (OCT), miniscope, multiphoton fluorescence (2PF & 3PF), adaptive optics, harmonic generation, and Raman microscopy for understanding pathomechanism of brain disorders and pathological and physiological changes associated with neurodegenerative diseases. Also, the book presents conventional imaging modalities, including Magnetic Resonance Imaging (MRI), for delineating underlying mechanisms and precise early diagnosis of neurological disorders. This book is a useful resource for neuroscientists and researchers working in biomedical engineering and optics.