Note: Front Cover -- Methods in Cell Biology -- Copyright Page -- Contents -- Contributors -- Preface -- Chapter 1: Autofluorescent Proteins -- I. History -- II. Variants -- A. Structure -- B. Stability, Folding, and Multimerization -- C. Spectra and Photophysical Dynamics -- III. Practical Considerations -- IV. Advanced FP Applications -- A. Multiple Labeling -- B. Dynamic Imaging -- C. Protein-Protein Interactions -- V. Future Directions -- References -- Chapter 2: Functional Fusion Proteins by Random Transposon-Based GFP Insertion -- I. Introduction -- II. Rationale -- III. Methods -- A. PCR Amplification of the Transposon -- B. The Transposition Reaction -- C. Transformation Requirements and Troubleshooting -- D. E.coli Colony Selection and Growth in a 96-Well Format -- E. Backing Up the Experiment: Making 10% Glycerol Stocks -- F. 96-Well Mini-Preparation Purification of Plasmid DNA -- G. Preparation of HEK 293 Cells -- H. Transient Transfection of HEK 293 Cells in a 96-Well Format -- I. Screening Live HEK 293 Cells for GEP Fluorescence -- J. Removing the Selection Cassette with Restriction Digestion and Re-Ligation -- IV. Materials -- A. PCR Amplification of the Transposon -- B. The Transposition Reaction -- C. Transformation Requirements and Troubleshooting -- D. E.coli Colony Selection and Growth in a 96-Well Format -- E. Backing Up the Experiment: Making 10% Glycerol Stocks -- F. 96-Well-Mini-Preparation Purification of Plasmid DNA -- G. Preparation of HEK 293 Cells -- H. Transient Transfection of HEK 293 Cells in a 96-Well Format -- I. Screening Live HEK 293 Cells for GEP Fluorescence -- J. Removing the Selection Cassette with Restriction Digestion and Re-Ligation -- V. Discussion -- Acknowledgments -- References -- Chapter 3: Fluorescent Proteins for Photoactivation Experiments -- I. Why Use a Fluorescent Protein?. , II. Why Use a Photoactivatable Fluorescent Protein? -- III. Survey of Photoactivatable Fluorescent Proteins -- A. Photoactivatable Fluorescent Proteins: Aequorea victoria GFP -- B. Photoactivatable Fluorescent Proteins: DsRed Fluorescent Protein -- C. Photoactivatable Fluorescent Proteins: Green-to-Red Photoconversions -- D. Photoactivatable Fluorescent Proteins: Cyan-to-Green Photoconversion -- E. Photoactivatable Fluorescent Proteins: Reversible -- IV. Uses of Photoactivatable Fluorescent Proteins -- A. Protein Dynamics -- B. Fluorescence Pulse-Labeling. -- C. Photoquenching Fluorescence Resonance Energy Transfer -- D. Photoactivated Localization Microscopy -- V. Future Directions of Photoactivatable Fluorescent Proteins -- References -- Chapter 4: Design and Optimization of Genetically Encoded Fluorescent Biosensors: GTPase Biosensors -- I. Introduction -- II. Background: Factors Influencing FRET Efficiency -- III. Design and Cloning of Biosensors -- IV. Validation of the Biosensor in Cell Suspensions -- A. Expression in HEK293T Cells for Assay of Biosensors in Cell Suspension -- B. Expression of the Biosensor in Cells -- V. Microscopy and Imaging Considerations -- VI. Conclusion -- VII. Appendix I -- A. DNA Sequence for the pTriEX-4-Biosensor Construct -- VIII. Appendix II -- A. Media Formulation for Ham's F-12K Phenol Red-Free -- References -- Chapter 5: Fast 4D Microscopy -- I. Introduction -- II. Fast 4D Imaging: Definition, Interest, and Limits -- III. Points to Consider Before Working with Fast 4D Imaging Systems -- A. Imaging Modes and Combination with Other Functionalities into a Multifunctional System -- B. Keeping the Sampled Volume Immobile and Test for It -- C. The Impact of Optical Blur, Noise, Aberrations, and Calibration Defects -- D. Setting Up a Rapid 4D Acquisition -- IV. Conclusions -- Acknowledgments -- References. , Chapter 6: Single-Molecule Imaging of Fluorescent Proteins -- I. Introduction -- II. Instrumentation -- III. Fluorophores -- IV. Reducing Protein Expression Levels -- V. Biological Preparations -- VI. Data Analysis and Interpretation -- VII. Future Prospects -- References -- Chapter 7: Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins -- I. Introduction -- II. Counting Kinetochore Protein Numbers in Budding Yeast -- A. Optimal Fluorescent Proteins -- III. Sample Preparation -- IV. Microscope and Image Acquisition System -- V. Measurement of Fluorescence Signal -- A. Characterization of the Point Spread Function of the Objective -- B. Characterization of Fluorescence Intensity Distribution for a Kinetochore Cluster and Signal Measurement -- VI. Validation of Measurement Method -- VII. Results -- VIII. Discussion -- A. The Choice of Calibration Standards for Quantitative Fluorescence Microscopy -- B. Counting Protein Numbers from Volumes Larger than the Diffraction Limit -- C. Sources of Error in Fluorescence Signal Measurement -- IX. Conclusions -- Acknowledgments -- References -- Chapter 8: Fluorescent Protein Applications in Plants -- I. Introduction -- II. Expression and Function of FPs in Plants -- A. Gene Expression -- B. Biosensors -- C. Assessing Function -- III. Imaging -- A. Protocol for 4D Imaging -- IV. Advanced Techniques -- A. Spectral Imaging -- B. Fluorescence Lifetime Imaging -- C. Fluorescence Correlation Spectroscopy -- V. Summary -- Acknowledgments -- References -- Chapter 9: Expression and Imaging of Fluorescent Proteins in the C. elegans Gonad and Early Embryo -- I. Introduction -- A. The Caenorhabditis elegans Gonad and Early Embryo: A Model System for Cell and Developmental Biology. , B. Quantitative Imaging-Based Assays Capitalize on the Rapid, Invariant Early Embryonic Cell Divisions -- II. Fluorescent Proteins in the C. elegans Gonad and Early Embryo -- A. Fluorescent Proteins Commonly Used in C. elegans -- B. Engineering New Fluorescent Proteins for Expression in the C. elegans Gonad/Early Embryo: The mCherry Experience -- III. Transgene Expression in the C. elegans Germline: Breaking the Silence -- A. Promoter and 3' UTR Choice -- B. Currently Available Vectors for Expression of Fluorescent Proteins in the Germline -- IV. Constructing Fluorescent Worm Lines -- A. Integration of Constructs by Ballistic Bombardment -- B. Making Dual/Triple Fluorescent Marker Lines by Mating -- C. Benefits and Challenges with Multimarker Lines -- V. Using Fluorescent Worm Strains -- A. Confirming Functionality of Transgenes -- B. Available Worm Strains for Imaging in the Gonad and Early Embryo -- C. Practical Techniques for Gonad/Embryo Imaging: Specimen Mounting and Drug Treatments -- D. Guidelines for Live Imaging of C. elegans Embryos -- VI. Summary -- Acknowledgments -- Appendix -- Media and Supplements -- Bombardment reagents -- References -- Chapter 10: Fluorescent Proteins in Zebrafish Cell and Developmental Biology -- I. Introduction -- II. Zebrafish Kinesin Genes in Early Development: A Cytokinetic Role for zMklp1 -- A. Cloning and Sequence Analysis of Zebrafish Kinesin cDNAs -- B. Engineering of Expression Constructs That Encode GFP-Tagged Wild-Type and Mutant zMklp1s -- C. In Vitro Synthesis of Capped GFP-Mklp1 mRNAs and Embryo Microinjection -- D. Results -- E. Future Applications and Improvements -- III. Cell-Specific, Laser-Induced Transgene Expression in the Zebrafish Embryo: The Sema3a1 Gene in Axonal Guidance -- A. Generation of sema3a1 Transgenic Zebrafish -- B. Laser Induction of Transgene Expression. , C. Detection of Axons and EGFP-Sema3a1-Myc by Immunohistochemistry -- D. Results -- E. Future Applications and Improvements -- IV. Transgenic Zebrafish Models of Myc-Induced T-Cell Acute Lymphoblastic Leukemia -- A. Generation of Zebrafish Containing the rag2:loxP-dsRED2-loxP-EGFP-mMycTransgene -- B. Activation of the Conditional mMyc Transgene by Injection of Cre Recombinase RNA and Analysis of Leukemic Cells -- C. Results -- D. Future Applications and Improvements -- V. Summary -- Acknowledgments -- References -- Chapter 11: Identifying and Quantitating Neural Stem and Progenitor Cells in the Adult Brain -- I. Introduction -- A. Neural Stem and Progenitor Cells and Neuronal Differentiation Cascade in the Adult Brain -- B. Adult Neurogenesis Is a Dynamic Process -- C. Identification and Quantification of Newborn Cells in the Adult Brain -- D. Nestin Marks Neural Stem and Progenitor Cells -- E. Transgenic Reporter Lines for Visualizing Neural Stem and Progenitor Cells -- F. Using Transgenic Reporter Lines to Dissect Neuronal Differentiation Cascade in the DG -- G. Using Reporter Lines to Quantify Neural Stem and Progenitor Cells -- II. Protocol I: Immunofluorescence Microscopy of Nestin-GFP and Nestin-CFPnuc Cells -- A. Perfusion -- B. Postfixation -- C. Sectioning -- D. Fluorescence Immunostaining -- E. BrdU Labeling -- F. Triple Labeling -- III. Protocol II: The Use of Confocal Stereology to Quantify Changes in Defined Classes of Neuronal Precursors -- A. The Fractionator Method -- B. The Optical Disector -- IV. Protocol III: Electron Microscopy of Nestin-GFP/CFPnuc Cells -- A. Perfusion -- B. Postfixation -- C. Sectioning -- D. 3,3'-Diaminobenzidine Immunostaining -- E. Contrasting and Dehydration -- F. Mounting and Embedding -- References -- Chapter 12: Using Fluorescent Proteins to Study mRNA Trafficking in Living Cells -- I. Introduction. , II. The MS2-GFP System.

Note: Front Cover -- Methods in Cell Biology -- Copyright Page -- Contents -- Contributors -- Preface -- Chapter I. Biophysics of the Green Fluorescent Protein -- I. Introduction -- II. Protein Folding and the Generation of This Chromophore -- III. The Biophysics of the Fluorescence of GTP -- IV. Resonance Energy Transfer Involving GFP -- V. Summary -- References -- Chapter 2. Understanding Structure-Function Relationships in the Aequorea Victoria Green Fluorescent Protein -- I. Introduction -- II. Structure -- III. Chromophore Formation -- IV. Effects of Mutations on the Spectroscopic Properties of GFP -- V. Effects of Mutations That Improve Thermosensitivity -- VI. The Development of Enhanced Mutants -- References -- Chapter 3. Quantitative Imaging of the Green Fluorescent Protein (GFP) -- I. Introduction -- II. Factors That Influence/Limit Quantitation of GFP in Fluorescence Microscopy -- III. Applications of LSCM for Quantitative Imaging of GFP -- IV. Preparation of Purified GFP Samples -- References -- Chapter 4. Single-Molecule Fluorescence Detection of Green Fluorescence Protein and Application to Single-Protein Dynamics -- I. Introduction -- II. Design Considerations for Fluorescence Microscopes for Single-Molecule Detection -- III. Characteristics of the Fluorescence from Single GFP Molecules -- IV. Advantages of Using GFP for Single-Molecule Detection -- V. GFP in Vitro and in Vivo Assays -- Appendix I. Details of the TIR Microscope -- Appendix II. Data Acquisition and Analysis -- References -- Chapter 5. Targeting GFP to Organelles -- I. Introduction -- II. Construction and Expression of the Organelle-Targeted GFP Chimeras -- III. Dynamic Monitoring of Organelle Structure with the Targeted GFPs -- IV. Expression in Primary Cultures -- V. Visualizing GFP Chimeras with Different Spectral Properties -- VI. Protocols -- References. , Chapter 6. Cytoskeletal Dynamics in Yeast -- I. Introduction -- II. Generating GFP Fusions -- III. Imaging Considerations for Yeast Cells -- IV. Time-Lapse Microscopy -- V. Results: Cytoskeletal GFP Fusion Proteins -- VI. Future -- References -- Chapter 7. Analysis of Nuclear Transport in Vivo -- I. Introduction -- II. Experimental Approaches and Protocols -- References -- Chapter 8. GFP Fusion Proteins as Probes for Cytology in Fission Yeast -- I. Introduction -- II. Expressing GFP Fusion Proteins -- III. Applications of Fusion Proteins -- References -- Chapter 9. GFP Variants for Multispectral Imaging of Living Cells -- 1. Introduction -- II. Green Fluorescent Protein Markers -- III. Imaging of Living Cells -- IV. Marking Different Cell Types in Arabidopsis -- V. Spectrally Distinct Fluorescent Proteins for Multichannel Confocal Microscopy -- VI. Summary -- References -- Chapter 10. GFP Fusions to a Microtubule Motor Protein to Visualize Meiotic and Mitotic Spindle Dynamics in Drosophila -- I. Introduction -- II. Labeling Strategies -- III. Imaging GFP -- IV. Applications of Ncd-GFP Imaging -- V. Perspectives -- References -- Chapter 11. GFP as a Cell and Developmental Marker i n the Drosophila Nervous System -- I. Introduction -- II. Targeted Expression of GFP in Drosophila -- III. Lines for Expression of GFP -- IV. Visualizing GFP Expression -- References -- Chapter 12. Using Time-Lapse Confocal Microscopy for Analysis of Centromere Dynamics in Human Cells -- I. Introduction -- II. GFP Fusion Proteins -- III. Microscopy -- IV. Analysis -- V. Summary -- Appendix: Handling Confocal Images on the Laboratory Computer -- References -- Chapter 13. Visualization of Largescale Chromatin Structure and Dynamics Using the lac Operator/lac Repressor Reporter System -- I. Introduction -- II. Overview of Methodology. , III. Construction of the lac Operator Repeat -- IV. Manipulation of the lac Operator Repeats -- V. Repressor-NLS and GFP-Repressor-NLS Constructs -- VI. Gene Amplification and Cell Cloning -- VII. Repressor Staining and Immunodetection of the lac Operator Repeat -- VIII. In Vivo Observation of GFP-Repressor Localization -- IX. Phototoxicity Issues -- X. Present Results and Future Directions -- References -- Chapter 14. Centrosome Dynamics in Living Cells -- I. Introduction -- II. Cloning and Expression of GFP-Pericentrin -- III. High-speed Microscopy -- IV. Image Restoration by an Improved Deconvolution Method -- V. Imaging Centrosomes -- VI. Postimaging Confirmation of Centrosome Integrity and Function -- References -- Chapter 15. Transfections of Primary Muscle Cell Cultures with Plasmids Coding for GFP Linked to Full-Length and Truncated Muscle Proteins -- I. Introduction -- II. Construction of GFP-Linked Muscle Proteins -- III. Preparation of Embryonic Avian Cardiomyocytes and Skeletal Muscle Myoblasts -- IV. Methods of Transfection of Cross-Striated Cells in Culture -- V. Transfection of Cross-Striated Muscle Cells with Full-Length cDNA for Sarcomeric Proteins -- VI. Microscopic Observations of Live Cells -- VII. Postprocessing of Transfected Cells -- VIII. Problems Encountered in Cells Transfected with GFP-Sarcomeric Proteins -- IX. Overview -- References -- Chapter 16. Monitoring the Dynamics and Mobility of Membrane Proteins Tagged with Green Fluorescent Protein -- I. Introduction -- II. Constructing and Expressing GFP Fusion Proteins: Strategies for Optimizing Brightness and Assessing Chimera Function -- III. Practical Guidelines for the Preparation and Imaging of GFP-Expressing Cells -- IV. Time-Lapse Imaging of GFP Chimeras: Critical Parameters -- V. Analysis of Time-Lapse Imaging Data. , VI. Relating GFP Chimera Fluorescence to Actual Numbers of GFP Molecules -- VII. Fluorescence Recovery after Photobleaching FRAP -- VIII. Qualitative FRAP Experiments -- IX. Quantitative FRAP -- X. Calculating D -- XI. Fluorescence Loss in Photobleaching (FLIP) Using a Confocal Microscope -- XII. Other Applications of Photobleaching -- References -- Chapter 17. Synchronous Real-Time Reporting of Multiple Cellular Events -- I. Introduction -- II. Green Fluorescent Protein -- III. Luciferase -- IV. Instrumentation and Techniques -- V. Future Directions -- References -- Chapter 18. Visualizing Protein Interactions in Living Cells Using Digitized GFP Imaging and FRET Microscopy -- I. Introduction -- II. The Theory of FRET -- III. Review of the FRET Literature -- IV. Why Use FRET Microscopy? -- V. Why Use the GFPs for FRET? -- VI. Some Considerations for the Use of GFPs in FRET Imaging -- VII. Some Considerations for Designing a FRET Imaging System -- VIII. The Practical Application of FRET to Visualize Protein- Protein Interactions -- IX. Overview and Conclusion -- References -- Chapter 19. Flow Cytometric Analysis and FACS Sorting of Cells Based on GFP Accumulation -- I. General Introduction -- II. Methods and Specific Applications -- III. Typical Results -- IV. Discussion and Conclusions -- References -- Chapter 20. GFP Biofluorescence: Imaging Gene Expression and Protein Dynamics in Living Cells -- I. Introduction -- II. Facilities -- III. Maintaining Cells -- IV. Imaging System -- V. Computer Systems -- VI. output -- VII. Conclusions -- References -- Index.

Note: Includes bibliographical references and index , ""Preface""; ""Contents""; ""A POCKET-SIZE INTRODUCTION""; ""THE BIG PICTURE - WITH EXAMPLES""; ""HOW TO SET THINGS UP? STUDY DESIGNS""; ""HOW OFTEN DOES IT HAPPEN? DISEASE FREQUENCY""; ""WHAT�S THE ANSWER? MEASURES OF EFFECT""; ""WHAT IS THE PUBLIC HEALTH IMPACT?""; ""IS THERE SOMETHING WRONG? VALIDITY AND BIAS""; ""WERE SUBJECTS CHOSEN BADLY? SELECTION BIAS""; ""ARE THE DATA CORRECT? INFORMATION BIAS""; ""OTHER FACTORS ACCOUNTED FOR? CONFOUNDING AND INTERACTION""; ""CONFOUNDING CAN BE CONFOUNDING - SEVERAL RISK FACTORS""; ""SIMPLE ANALYSES- 2x2 TABLES ARE NOT THAT SIMPLE"" , ""CONTROL - WHAT IT�S ALL ABOUT""""HOW TO DEAL WITH LOTS OF TABLES? STRATIFIED ANALYSIS""; ""MATCHING - SEEMS EASY, BUT NOT THAT EASY""; ""Index""