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Dedication; Foreword; On the Question of GPCR Oligomerization; References; Preface; Contents; Part I: Introduction; Chapter 1: Historical Perspectives: From Monomers to Dimers and Beyond, an Exciting Journey in the World of G Protein-Coupled Receptors; 1.1 Introduction; 1.2 The Birth of the Dimerization Concept; 1.3 Establishing the New Concept; 1.4 Oligomers Make the Picture More Complicated; 1.5 Receptor Homo- and Heteromers as New Pharmacological Targets; 1.6 Perspectives; References
Chapter 2: The Use of Spatial Intensity Distribution Analysis to Examine G Protein-Coupled Receptor Oligomerization2.1 Introduction; 2.1.1 SpIDA Procedure; 2.1.2 Choice of Fluorophore; 2.1.3 Selection of the Expression System; 2.1.4 Establishing Imaging Conditions for SpIDA; 2.1.4.1 Laser Power Intensity Measurement; 2.1.4.2 Laser Spot Beam Waist Radius Size; 2.1.4.3 Analog Detector Calibration; 2.1.4.4 Assessment of White Noise Level; 2.1.5 Laser Scanning Confocal Image Acquisition; 2.1.6 Spatial Intensity Distribution Analysis of Laser Scanning Confocal Images
2.1.6.1 Determining the Monomeric Quantal Brightness Value2.1.6.2 1 Population or 2 Population Mode; 2.1.6.3 When Does a QB Value Reflect Monomeric and When Dimeric/Oligomeric States?; 2.1.7 Determining the Quaternary Structure of GPCRs and How This May Be Affected by Ligand Binding; 2.1.8 Future Perspectives of SpIDA; References; Chapter 3: Advanced Microscopy Techniques; 3.1 Introduction; 3.2 FRET-Based Microscopy; 3.2.1 Definitions and the Principle of the Method; 3.2.2 Theoretical Basis of the Method; 3.2.3 Practical Implementation of FRET
3.2.3.1 General Requirements for Optical Microscopes Used in FRET Studies3.2.3.2 Two-Photon Absorption Optical Micro-spectroscopy; 3.2.3.3 Determination of FRET Efficiency from Optical Micro-Spectroscopy Data; 3.2.4 Theoretical Models Used for Information Extraction from Experimental Data; 3.2.4.1 Prediction of FRET Efficiencies for Various Oligomer Configurations; 3.2.4.2 Computation of Average FRET Efficiencies for Mixtures of Oligomers; 3.2.5 Experimental Applications of FRET; 3.2.5.1 FRET Spectrometry; 3.2.5.2 Statistical-Ensemble Approach to FRET
3.2.5.3 Combination Between the Two FRET Approaches3.3 Spatial Fluorescence Correlation Spectroscopy; 3.3.1 Principle of the Method; 3.3.1.1 Generalized Spatial Correlation Function; 3.3.1.2 Spatial Auto-Correlation; 3.3.1.3 Spatial Cross-Correlation and Colocalization; 3.3.2 Practical Implementation of ICCS; 3.3.3 Experimental Applications of Spatial Fluorescence Correlation Spectroscopy; 3.4 Temporal Fluorescence Correlation Spectroscopy; 3.4.1 Principle of the Method; 3.4.2 Theoretical Background of the Method; 3.4.2.1 Generalized Temporal Correlation Function
Chapter 2: The Use of Spatial Intensity Distribution Analysis to Examine G Protein-Coupled Receptor Oligomerization2.1 Introduction; 2.1.1 SpIDA Procedure; 2.1.2 Choice of Fluorophore; 2.1.3 Selection of the Expression System; 2.1.4 Establishing Imaging Conditions for SpIDA; 2.1.4.1 Laser Power Intensity Measurement; 2.1.4.2 Laser Spot Beam Waist Radius Size; 2.1.4.3 Analog Detector Calibration; 2.1.4.4 Assessment of White Noise Level; 2.1.5 Laser Scanning Confocal Image Acquisition; 2.1.6 Spatial Intensity Distribution Analysis of Laser Scanning Confocal Images
2.1.6.1 Determining the Monomeric Quantal Brightness Value2.1.6.2 1 Population or 2 Population Mode; 2.1.6.3 When Does a QB Value Reflect Monomeric and When Dimeric/Oligomeric States?; 2.1.7 Determining the Quaternary Structure of GPCRs and How This May Be Affected by Ligand Binding; 2.1.8 Future Perspectives of SpIDA; References; Chapter 3: Advanced Microscopy Techniques; 3.1 Introduction; 3.2 FRET-Based Microscopy; 3.2.1 Definitions and the Principle of the Method; 3.2.2 Theoretical Basis of the Method; 3.2.3 Practical Implementation of FRET
3.2.3.1 General Requirements for Optical Microscopes Used in FRET Studies3.2.3.2 Two-Photon Absorption Optical Micro-spectroscopy; 3.2.3.3 Determination of FRET Efficiency from Optical Micro-Spectroscopy Data; 3.2.4 Theoretical Models Used for Information Extraction from Experimental Data; 3.2.4.1 Prediction of FRET Efficiencies for Various Oligomer Configurations; 3.2.4.2 Computation of Average FRET Efficiencies for Mixtures of Oligomers; 3.2.5 Experimental Applications of FRET; 3.2.5.1 FRET Spectrometry; 3.2.5.2 Statistical-Ensemble Approach to FRET
3.2.5.3 Combination Between the Two FRET Approaches3.3 Spatial Fluorescence Correlation Spectroscopy; 3.3.1 Principle of the Method; 3.3.1.1 Generalized Spatial Correlation Function; 3.3.1.2 Spatial Auto-Correlation; 3.3.1.3 Spatial Cross-Correlation and Colocalization; 3.3.2 Practical Implementation of ICCS; 3.3.3 Experimental Applications of Spatial Fluorescence Correlation Spectroscopy; 3.4 Temporal Fluorescence Correlation Spectroscopy; 3.4.1 Principle of the Method; 3.4.2 Theoretical Background of the Method; 3.4.2.1 Generalized Temporal Correlation Function