1;Preface;5 2;Contents;8 3;Contributors;16 4;Considerations for Routine Imaging;21 4.1;Entering the Portal: Understanding the Digital Image Recorded Through a Microscope;22 4.1.1;1.1 Introduction;22 4.1.2;1.2 Historical Perspective;23 4.1.3;1.3 Digital Image Acquisition: Analog to Digital Conversion;23 4.1.4;1.4 Spatial Resolution in Digital Images;25 4.1.5;1.5 The Contrast Transfer Function;27 4.1.6;1.6 Image Brightness and Bit Depth;29 4.1.7;1.7 Image Histograms;30 4.1.8;1.8 Fundamental Properties of CCD Cameras;31 4.1.9;1.9 CCD Enhancing Technologies;35 4.1.10;1.10 CCD Performance Measures;36 4.1.11;1.11 Multidimensional Imaging;40 4.1.12;1.12 The Point-Spread Function;43 4.1.13;1.13 Digital Image Display and Storage;47 4.1.14;1.14 Imaging Modes in Optical Microscopy;48 4.1.15;1.15 Summary;58 4.1.16;1.16 Internet Resources;60 4.1.17;References;60 4.2;Quantitative Biological Image Analysis;63 4.2.1;2.1 Introduction;63 4.2.2;2.2 Definitions and Perspectives;64 4.2.3;2.3 Image Preprocessing;66 4.2.4;2.4 Advanced Processing for Image Analysis;75 4.2.5;2.5 Higher-Dimensional Data Visualization;81 4.2.6;2.6 Software Tools and Development;84 4.2.7;References;86 4.3;The Open Microscopy Environment: A Collaborative Data Modeling and Software Development Project for Biological Image Informatics;89 4.3.1;3.1 Introduction;89 4.3.2;3.2 OME Specifications and File Formats;92 4.3.3;3.3 OME Data Management and Analysis Software;95 4.3.4;3.4 Conclusions and Future Directions;108 4.3.5;References;108 4.4;Design and Function of a Light-Microscopy Facility;111 4.4.1;4.1 Introduction;111 4.4.2;4.2 Users;113 4.4.3;4.3 Staff;114 4.4.4;4.4 Equipment;116 4.4.5;4.5 Organization;121 4.4.6;4.6 Summary;130 4.4.7;References;131 5;Advanced Methods and Concepts;132 5.1;Quantitative Colocalisation Imaging: Concepts, Measurements, and Pitfalls;133 5.1.1;5.1 Introduction;133 5.1.2;5.2 Quantifying Colocalisation;153 5.1.3;5.3 Conclusions;166 5.1.4;References;167 5.2;Quantitative FRET Microscopy of Live Cells;172 5.2.1;6.1 Introduction;172 5.2.2;6.2 Introductory Physics of FRET;173 5.2.3;6.3 Manifestations of FRET in Fluorescence Signals;175 5.2.4;6.4 Molecular Interaction Mechanisms That Can Be Observed by FRET;178 5.2.5;6.5 Measuring Fluorescence Signals in the Microscope;180 5.2.6;6.6 Methods for FRET Microscopy;182 5.2.7;6.7 Fluorescence Lifetime Imaging Microscopy for FRET;190 5.2.8;6.8 Data Display and Interpretation;191 5.2.9;6.9 FRET-Based Biosensors;192 5.2.10;6.10 FRET Microscopy for Analyzing Interaction Networks in Live Cells;193 5.2.11;6.11 Conclusion;195 5.2.12;References;195 5.3;Fluorescence Photobleaching and Fluorescence Correlation Spectroscopy: Two Complementary Technologies To Study Molecular Dynamics in Living Cells;197 5.3.1;7.1 Introduction;197 5.3.2;7.2 Fundamentals;203 5.3.3;7.3 How To Perform a FRAP Experiment;210 5.3.4;7.4 How To Perform an FCS Experiment;219 5.3.5;7.5 How To Perform a CP Experiment;231 5.3.6;7.6 Quantitative Treatment;235 5.3.7;7.7 Conclusion;241 5.3.8;References;241 5.4;Single Fluorescent Molecule Tracking in Live Cells;248 5.4.1;8.1 Introduction;248 5.4.2;8.2 Tracking of Single Chromosomal Loci;249 5.4.3;8.3 Single-Molecule Tracking of mRNA;260 5.4.4;8.4 Single-Particle Tracking for Membrane Proteins;266 5.4.5;8.5 Tracking Analysis and Image Processing of Data from Particle Tracking in Living Cells;271 5.4.6;8.6 Conclusion;271 5.4.7;8.7 Protocols for Laboratory Use;272 5.4.8;References;274 5.5;From Live-Cell Microscopy to Molecular Mechanisms: Deciphering the Functions of Kinetochore Proteins;277 5.5.1;9.1 Introduction;277 5.5.2;9.2 Biological Problem: Deciphering the Functions of Kinetochore Proteins;280 5.5.3;9.3 Experimental Design;281 5.5.4;9.4 Extraction of Dynamics from Images;285 5.5.5;9.5 Characterization of Dynamics;288 5.5.6;9.6 Quantitative Genetics of the Yeast Kinetochore;294 5.5.7;9.7 Conclusion;296 5.5.8;References;296 6;Cutting Edge Applications & Utilities;298 6.1;Towards Imaging the Dynamics of P