Note: Intro -- Single Molecule Spectroscopy in Chemistry, Physics and Biology -- Part I Introductory Lecture: Molecular Dynamics of Single Molecules -- 1 How Biomolecular Motors Work: Synergy Between Single Molecule Experiments and Single Molecule Simulations -- Acknowledgments -- References -- Part II Detection of Single Molecules and Single Molecule Processes -- 2 Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances -- 2.1 Introduction -- 2.2 Early Steps: Statistical Fine Structure in Inhomogeneous Lines -- 2.3 A Scaling Argument Led the way to the First Single-Molecule Detection and Spectroscopy -- 2.4 Selected Low-Temperature Milestones -- 2.4.1 Imaging Single Molecules in Frequency and Space -- 2.4.2 Observation of Spectral Diffusion of a Single Molecule -- 2.4.3 Optical Switching of Single Molecules -- 2.4.4 Photon Antibunching and Magnetic Resonance of Single Molecular Spins -- 2.4.5 Single Molecules Interacting with Novel Optical Fields -- 2.5 Selected Room Temperature Milestones -- 2.5.1 Blinking and Switching for Single Green Fluorescent Proteins -- 2.5.2 FRET for a Dual-GFP Construct -- 2.5.3 imaging in cells -- Diffusion -- Imaging of Single Molecules in Bacteria -- Superresolution Imaging by Treadmilling -- 2.5.4 New Photoactivatable and Photoswitchable Single-Molecule Fluorophores -- 2.5.5 Trapping Single Molecules in Aqueous Solution: ABEL Trap -- 2.6 Summary -- Acknowledgements -- References -- 3 Single Molecules as Optical Probes for Structure and Dynamics -- 3.1 Introduction -- 3.1.1 Optical Signals from Single Nano-Objects in the Far Field -- 3.1.2 Signal-to-Noise Ratio -- Direct Absorption -- Fluorescence -- Photothermal Detection -- 3.2 Examples of Nanoscale Probing -- 3.2.1 Blinking -- 3.2.2 Gold Nanoparticles -- 3.2.3 Cryogenic Single-Molecule Spectroscopy -- 3.2.4 Supercooled Liquids. , The Glass Transition -- Liquid-Like Pockets and Solid-Like Structures -- Rheology -- Open Questions -- 3.3 Outlook and Conclusion -- Acknowledgements -- References -- 4 FCS and Single Molecule Spectroscopy -- 4.1 Introduction -- 4.2 Fluorescence Correlation Spectroscopy -- 4.2.1 Kinetics of the Excited State and Rotational Correlations -- 4.2.2 Rotational Diffusion -- 4.3 Excited States Lifetime and Antibunching -- 4.4 FCS in Confocal Volumes -- 4.5 Confocal Single Molecule Detection and Single Molecule Imaging -- 4.6 Single Molecule Dynamics -- 4.7 Non-Ergodic Behavior -- 4.8 Triplet Kinetics -- 4.9 Relaxation Kinetics -- 4.10 Single Enzyme Molecule Kinetics -- 4.11 Stretched Exponentials -- 4.12 Memory Landscapes -- 4.13 Single Molecules in the Electric Field -- 4.14 FCS Cross Correlation and Applications -- 4.15 Single Molecule Detection in Single Cells -- 4.16 Outlook -- Acknowledgement -- References -- Part III Fluorescence-Correlation Spectroscopy -- 5 Single-Molecule Spectroscopy Illuminating the Molecular Dynamics of Life -- 5.1 Fluorescence Correlation Spectroscopy in Its Historical Context -- 5.2 Multiphoton Microscope and Photophysical Technologies Probing Molecular Dynamics of Life -- 5.3 Single-Molecule Diffusion Anomalies (and Membrane Structures) -- 5.4 Single-Molecule Spectroscopy in Neuroscience -- 5.5 FCS Protein Structure Fluctuations in Neurodegenerative Disease -- 5.6 Molecular Transcription Factor Activation in the Cell Nucleus -- 5.7 Efficient DNA Sequencing by Single-Molecule Spectroscopy -- 5.8 A Medical Future for Our ``Single-Molecule Spectroscopy Illuminating the Molecular Dynamics of Life'' -- References -- 6 Chemical Fluxes in Cellular Steady States Measured by Fluorescence-Correlation Spectroscopy -- 6.1 Introduction -- 6.2 Steady States of Nonequilibrium Chemical Reaction Networks. , 6.3 Noise and Fluctuations in Biological Reaction Networks -- 6.4 Experimental Characterization of Steady State Fluxes -- 6.5 Summary and Conclusions -- References -- 7 In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy -- 7.1 Fluorescence Correlation and Cross-Correlation Spectroscopy -- 7.2 Two-Photon Scanning FCS in C. elegans embryos -- 7.2.1 Scanning FCS -- 7.2.2 FCS in the Cytoplasm -- 7.2.3 sFCS in the Cortex -- 7.2.4 Discussion -- 7.3 FCS and FCCS Elucidate the RNA Interference Pathway -- 7.3.1 FCS and FCCS Methods -- 7.3.2 FCS Results -- 7.3.3 FCCS Results -- 7.3.4 Discussion -- References -- 8 Fluorescence Flicker as a Read-Out in FCS: Principles, Applications, and Further Developments -- 8.1 Introduction -- 8.2 Monitoring of Ion Concentration and Exchange -- 8.3 Photo-Induced Transient States -- 8.4 Conclusions -- Acknowledgements -- References -- Part IV Quantum Dots and Single Molecule Behaviour -- 9 Development of Nanocrystal Molecules for Plasmon Rulers and Single Molecule Biological Imaging -- References -- 10 Size-Minimized Quantum Dots for Molecular and Cellular Imaging -- 10.1 Introduction -- 10.2 Results and Discussion -- 10.2.1 Molar Capping Ratio -- 10.2.2 Minimum Size -- 10.2.3 Stability -- 10.2.4 Size Comparison with Proteins -- 10.2.5 Super-Resolution Imaging -- 10.3 Experimental Section -- 10.3.1 Polymer Synthesis -- 10.3.2 Determination of Reactive Amines and Thiols -- 10.3.3 Ligand Exchange with Thioglycerol -- 10.3.4 Coating with Multidentate Polymer Ligands -- 10.3.5 Calculation of Molar Capping Ratio -- 10.3.6 Calculation of Surface Atoms Per Nanocrystal -- Acknowledgements -- References -- 11 Mapping Transcription Factors on Extended DNA: A Single Molecule Approach -- 11.1 Introduction -- 11.2 Concept of Single Molecule ChIP: Seeing is Believing -- 11.3 Experiment and Results. , 11.4 Discussion and Outlook -- 11.5 Methods -- 11.5.1 Protein Expression and Purification -- 11.5.2 Binding and Labeling Reaction -- 11.5.3 Surface Preparation -- Acknowledgements -- References -- Part V Molecular Motion of Contractile Elements and Polymer Formation -- 12 Single-Molecule Measurement, a Tool for Exploring the Dynamic Mechanism of Biomolecules -- 12.1 Introduction: Fluctuation and Single-Molecule Measurements -- 12.2 Biased Brownian Movement of Muscle Myosin -- 12.3 Forward and Backward Step Movement of Kinesin -- 12.4 Directional Movement of Processive Myosin -- 12.5 Conformational Fluctuation of Actin and Activation of Myosin Motility -- 12.6 Biased Brownian Mechanism of Myosin Movement and Muscle Contraction -- 12.7 Multiple Conformations of Ras and Switching Signals -- 12.8 Multiple States of Signaling Proteins in Living Cells -- 12.9 Kinetic Heterogeneity of Cell Signaling Processes in Living Cells -- 12.10 Direct Determination of the Input-Output Relation in Protein Molecules -- 12.11 Future Perspective -- References -- 13 Viral DNA Packaging: One Step at a Time -- 13.1 Introduction -- 13.2 The Bacteriophage Packaging Motor -- 13.3 A Single Molecule Packaging Assay -- 13.4 Single-Molecule Mechanochemistry -- 13.5 Hints of Coordination Among the ATPases -- 13.6 Resolving the Discrete Steps of the Packaging Motor -- 13.7 DNA is Packaged in 10-bp Increments -- 13.8 Multiple ATPs Bind Before each 10-bp Step -- 13.9 Packaging Occurs in Bursts of Four 2.5-bp Steps -- 13.10 Inter-Subunit Coordination in the Packaging Motor -- 13.11 Summary and Conclusions -- Acknowledgments -- References -- 14 Chemo-Mechanical Coupling in the Rotary Molecular Motor F1-ATPase -- 14.1 Introduction -- 14.2 Rotation Scheme -- 14.2.1 Data Selection -- 14.2.2 120 Step Per ATP -- 14.2.3 Substeps -- 14.2.4 Direction of Rotation -- 14.3 Chemo-Mechanical Coupling. , 14.3.1 ATP Binding -- 14.3.2 Hydrolysis of ATP -- 14.3.3 Pi Release -- 14.3.4 ADP Release -- 14.3.5 Site Occupancy -- 14.4 Energetics of Coupling -- 14.4.1 Pi Release -- 14.4.2 Power Stroke vs. Diffusion and Catch -- 14.4.3 Chemo-Mechanical Coupling by Induced Fit and Unfit -- 14.5 Structural Basis of Rotation -- 14.5.1 Crystal Structures -- 14.5.2 Axle-Less Constructs -- 14.6 Remaining Tasks -- Acknowledgements -- References -- Part VI Force and Multiparameter Spectroscopy on Functional Active Proteins -- 15 Mechanoenzymatics and Nanoassembly of Single Molecules -- 15.1 Introduction -- 15.2 The Molecular Force Sensor Titin Kinase -- 15.3 One-by-One Assembly of Single Molecules -- 15.4 Concluding Remarks -- Acknowledgments -- References -- 16 Single Cell Physiology -- 16.1 Introduction -- 16.2 Caged Molecules -- 16.3 Optical Control in Neurophysiology -- 16.4 Optical Control of Gene Expression -- 16.5 Optical Control of RNA Expression -- 16.6 Optical Control of an Endogenous Morphogen: Retinoic Acid -- 16.7 Optical Control of Protein Activity -- Acknowledgements -- References -- 17 Force-Clamp Spectroscopy of Single Proteins -- 17.1 The Importance of Polyprotein Engineering -- 17.2 Force-Clamp Spectroscopy of Polyproteins -- 17.3 Force-Clamp Spectroscopy Measures the Distance to the Transition State x -- 17.4 Force Dependency of Protein Unfolding -- 17.5 Molecular Interpretation of x in Protein Unfolding -- 17.6 The Force Dependency of Chemical Reactions -- 17.7 Molecular Interpretation of x in Chemical Reactions -- 17.8 A Statistical Dynamics View of Protein Folding -- 17.9 The Force-Quench Experiment -- 17.10 Summary -- References -- 18 Unraveling the Secrets of Bacterial Adhesion Organelles Using Single-Molecule Force Spectroscopy -- 18.1 Introduction -- 18.2 Instrumentation, Procedures, and Typical Force-vs.-Elongation Response of Pili. , 18.2.1 Force-Measuring Optical Tweezers - Instrumentation.