This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.
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1. Fluorescent Proteins 2. Functional Fusion Proteins by Random Transposon-Based GFP Insertion 3. Fluorescent Proteins for Photoactivation Experiments 4. Design and Optimization of Genetically Encoded Fluorescent Biosensors: Gtpase Biosensors 5. Fast 4D Microscopy 6. Single molecule imaging of fluorescent proteins 7. Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins 8. Fluorescent Protein Applications in Plants 9. Expression and Imaging of Fluorescent Proteins in the C. elegans Gonad and Early Embryo 10. Fluorescent Proteins in Zebrafish Cell and Developmental Biology 11. Identifying and Quantitating Neural Stem and Progenitor Cells in the Adult Brain 12. Using Fluorescent Proteins to Study mRNA Trafficking in Living Cells 13. Visualising mRNA Localisation and Local Protein Translation in Neurons 14. Quantitative FRAP in Analysis of Molecular Binding Dynamics In Vivo 15. Quantitative and Qualitative Analysis of Plant Membrane Traffic Using Fluorescent Proteins 16. Engineering FRET Constructs Using CFP and YFP 17. Fluorescence Anisotropy Imaging Microscopy (FAIM) for homo-FRET in Living Cells 18. FRET by Fluorescence Polarization Microscopy 19. Bimolecular Fluorescence Complementation Visualization of Molecular Interactions in Living Cells 20. Protein-protein interactions determined by fluorescence correlation spectroscopy 21. Recent Advances on In Vivo Imaging with Fluorescent Proteins 22. Computational Processing and Analysis of Dynamic Image Data 23. Automated Classification of Mitotic Phenotypes of Human Cells Using Fluorescent Proteins 24. Open Tools for Storage and Management of Quantitative Image Data