Multiphoton Microscopy And Fluorescence Lifetime Imaging

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

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.

During the past two decades, there has been an increasing appreciation of the significant value that lifetime-based techniques can add to biomedical studies and applications of fluorescence. Bringing together perspectives of different research communities, Fluorescence Lifetime Spectroscopy and Imaging: Principles and Applications in Biomedical Diagnostics explores the remarkable advances in time-resolved fluorescence techniques and their role in a wide range of biological and clinical applications. Broadly accessible, the book captures the state-of-the-art of fluorescence lifetime metrology and imaging and provides current perspectives on their applications to biomedical studies of intact tissues and medical diagnosis. The text introduces these techniques within the wider context of fluorescence spectroscopy and describes basic principles underlying current instrumentation for fluorescence lifetime imaging and metrology (FLIM). It also covers the wide range of methods, including single channel (point) spectroscopy, fluorescence lifetime imaging microscopy, and single- and multi-photon excitation. Edited by pioneers in this field, with contributions from leading experts, the book includes an overview of complementary techniques that help researchers beginning FLIM research. It offers a comprehensive treatment of fundamental principles, instrumentation, analytical methods, and applications. It also provides an overview of the label-free contrast available from lifetime measurements of tissue autofluorescence and the prospects for exploiting this for clinical applications and biomedical research including drug discovery.

In the last decade multiphoton excitation microscopy has emerged as an important technique with ever increasing numbers of significant applications in the fields of biology, chemistry, physics, and medicine. This volume contains key papers on the following topics: developments of nonlinear optical spectroscopy and nonlinear scanning microscopy (SHG, CARS); theory and techniques of multiphoton excitation microscopy; development of laser sources; single-molecule studies; and applications to biology, cell biology, embryology and developmental biology, neuroscience, dermatology, and optical biopsy. A comprehensive bibliography follows the reprinted papers.

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.

During the past two decades, there has been an increasing appreciation of the significant value that lifetime-based techniques can add to biomedical studies and applications of fluorescence. Bringing together perspectives of different research communities, Fluorescence Lifetime Spectroscopy and Imaging: Principles and Applications in Biomedical Diagnostics explores the remarkable advances in time-resolved fluorescence techniques and their role in a wide range of biological and clinical applications. Broadly accessible, the book captures the state-of-the-art of fluorescence lifetime metrology and imaging and provides current perspectives on their applications to biomedical studies of intact tissues and medical diagnosis. The text introduces these techniques within the wider context of fluorescence spectroscopy and describes basic principles underlying current instrumentation for fluorescence lifetime imaging and metrology (FLIM). It also covers the wide range of methods, including single channel (point) spectroscopy, fluorescence lifetime imaging microscopy, and single- and multi-photon excitation. Edited by pioneers in this field, with contributions from leading experts, the book includes an overview of complementary techniques that help researchers beginning FLIM research. It offers a comprehensive treatment of fundamental principles, instrumentation, analytical methods, and applications. It also provides an overview of the label-free contrast available from lifetime measurements of tissue autofluorescence and the prospects for exploiting this for clinical applications and biomedical research including drug discovery.