1st Edition
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.
INTRODUCTION, MICROSCOPY, FLUOROPHORES
Fluorescence Lifetime Resolved Imaging-What, Why, How: A Prologue, Robert M. Clegg, University of Illinois at Urbana-Champaign
Principles of Fluorescence for Quantitative Fluorescence Microscopy, Neil Anthony, Peng Guo and Keith Berland, Emory University
Visible Fluorescent Proteins for FRET-FLIM, Richard N. Day, Indiana University School of Medicine
INSTRUMENTATION
Wide-field Fluorescence Lifetime Imaging Microscopy using a Gated Image Intensifier Camera, Yuansheng Sun, James N. Demas and Ammasi Periasamy, University of Virginia
Frequency-Domain FLIM, Bryan Q. Spring and Robert M. Clegg, University of Illinois at Urbana-Champaign
Laser Scanning Confocal FLIM Microscopy, Hans Gerritsen, Arjen Bader and Sasha Agronskaia, University of Utrecht, Netherlands
Multiphoton Fluorescence Lifetime Imaging at the Dawn of Clinical Application, Karsten König and Aisada Uchugonova, Saarland University, Germany
FLIM microscopy with Streak Camera, V. Krishnan Ramanujan , Javier A. Jo, Ravi Ranjan and Brian A. Herman, Cedars-Sinai Medical Center, LA and University of Texas at San Antonio
Spectrally Resolved Fluorescence Lifetime Imaging Microscopy: SLIM/mwFLIM, Christoph Biskup, Frank Dolp, Birgit Hoffmann, Klaus Benndorf, and Angelika Rück, Universitätsklinikum Jena and University of Ulm, Germany
Time Resolved Fluorescence Anisotropy, Steven S. Vogel, Christopher Thaler, Paul S. Blank and Srinagesh V. Koushik, National Institutes of Health
DATA ANALYSIS
General Concerns of FLIM Data Representation and Analysis and Frequency Domain Model-Free Analysis, Yi-Chun Chen, Bryan Q. Spring, Chttanon Buranachai, Bianca Tong, George Malachowski and Robert M. Clegg, University of Illinois at Urbana-Champaign
Non-linear Curve Fitting Methods for Time-Resolved Data Analysis, Ignacy Gryczynski, Rafal Luchowski, Shashank Bharill, Julian Borejdo, and Zygmunt Gryczynski, University of North Texas Health Sciences Center
Global Analysis of Frequency Domain FLIM Data, Hernan E. Grecco and Peter J. Verveer, Max Planck Institute of molecular Physiology, Germany
APPLICATIONS
FLIM Applications in the Biomedical Sciences, Ammasi Periasamy, University of Virginia and Robert Clegg, University of Illinois at Urbana-Champaign
Biography
Ammasi Periasamy, University of Virginia, Charlottesville, USA
Robert M. Clegg, University of Illinois, Urbana-Champaign, USA
"…this book represents a clear presentation of the theory, instrumentation, and data analysis of FLI microscopy and is enhanced by the critical discussions of assumptions, limitations and confounding elements… ."
—Barry R. Masters, MIT, Microscopy and Microanalysis"…an excellent and comprehensive treatment of FRET in biology investigation. It has a good balance of basic theory and cutting-edge development in both techniques and applications."
—Peter So, Ph.D., MIT"An amazingly comprehensive reference—I can’t imagine anyone looking for information there and not finding what they need. It is quite essential for any lab doing FLIM."
—Guy Cox, University of Sydney, Australia
