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Fluorescence microscopy Elektronische Ressource from principles to biological applications edited by Ulrich Kubitscheck

Contributor(s): Kubitscheck, Ulrich [HerausgeberIn]Material type: TextTextLanguage: English Publisher: Weinheim Wiley-VCH 2017Edition: Second editionDescription: 1 Online-Ressource (507 pages)Content type: Text Media type: Computermedien Carrier type: Online-RessourceISBN: 9783527687725; 9783527687749; 9783527687756; 9783527687732; 9783527338375 (print)Subject(s): Fluoreszenzmikroskopie | Fluorescence microscopyAdditional physical formats: Print version: Fluorescence Microscopy : From Principles to Biological ApplicationsDDC classification: 502.82 Other classification: 42.03 | 33.38 | WC 2900 Online resources: Volltext Summary: Cover -- Title Page -- Copyright -- Contents -- List of Contributors -- Preface -- Chapter 1 Introduction to Optics -- 1.1 A Short History of Theories about Light -- 1.2 Properties of Light Waves -- 1.2.1 An Experiment on Interference -- 1.2.2 Physical Description of Light Waves -- 1.3 Four Effects of Interference -- 1.3.1 Diffraction -- 1.3.2 The Refractive Index -- 1.3.3 Refraction -- 1.3.4 Reflection -- 1.3.5 Light Waves and Light Rays -- 1.4 Optical Elements -- 1.4.1 Lenses -- 1.4.2 Metallic Mirrors -- 1.4.3 Dielectric Mirrors -- 1.4.4 Filters -- 1.4.5 Chromatic Reflectors -- 1.5 Optical Aberrations -- References -- Chapter 2 Principles of Light Microscopy -- 2.1 Introduction -- 2.2 Construction of Light Microscopes -- 2.2.1 Components of Light Microscopes -- 2.2.2 Imaging Path -- 2.2.3 Magnification -- 2.2.4 Angular and Numerical Aperture -- 2.2.5 Field of View -- 2.2.6 Illumination Beam Path -- 2.2.6.1 Critical and Köhler Illumination -- 2.2.6.2 Bright-Field and Epi-Illumination -- 2.3 Wave Optics and Resolution -- 2.3.1 Wave Optical Description of the Imaging Process -- 2.3.2 The Airy Pattern -- 2.3.3 Point Spread Function and Optical Transfer Function -- 2.3.4 Lateral and Axial Resolution -- 2.3.4.1 Lateral Resolution Using Incoherent Light Sources -- 2.3.4.2 Lateral Resolution of Coherent Light Sources -- 2.3.4.3 Axial Resolution -- 2.3.5 Magnification and Resolution -- 2.3.6 Depth of Field and Depth of Focus -- 2.3.7 Over- and Undersampling -- 2.4 Apertures, Pupils, and Telecentricity -- 2.5 Microscope Objectives -- 2.5.1 Objective Lens Design -- 2.5.2 Light Collection Efficiency and Image Brightness -- 2.5.3 Objective Lens Classes -- 2.5.4 Immersion Media -- 2.5.5 Special Applications -- 2.6 Contrast -- 2.6.1 Dark Field -- 2.6.2 Phase Contrast -- 2.6.2.1 Frits Zernike's Experiments -- 2.6.2.2 Setup of a Phase-Contrast MicroscopeSummary: 2.6.2.3 Properties of Phase-Contrast Images -- 2.6.3 Interference Contrast -- 2.6.4 Advanced Topic: Differential Interference Contrast -- 2.6.4.1 Optical Setup of a DIC Microscope -- 2.6.4.2 Interpretation of DIC Images -- 2.6.4.3 Comparison between DIC and Phase Contrast -- 2.7 Summary -- Acknowledgments -- References -- Chapter 3 Fluorescence Microscopy -- 3.1 Contrast in Optical Microscopy -- 3.2 Physical Foundations of Fluorescence -- 3.2.1 What is Fluorescence? -- 3.2.2 Fluorescence Excitation and Emission Spectra -- 3.3 Features of Fluorescence Microscopy -- 3.3.1 Image Contrast -- 3.3.2 Specificity of Fluorescence Labeling -- 3.3.3 Sensitivity of Detection -- 3.4 A Fluorescence Microscope -- 3.4.1 Principle of Operation -- 3.4.2 Sources of Exciting Light -- 3.4.3 Optical Filters in a Fluorescence Microscope -- 3.4.4 Electronic Filters -- 3.4.5 Photodetectors for Fluorescence Microscopy -- 3.4.6 CCD or Charge-Coupled Device -- 3.4.7 Intensified CCD (ICCD) -- 3.4.8 Electron-Multiplying Charge-Coupled Device (EMCCD) -- 3.4.9 CMOS -- 3.4.10 Scientific CMOS (sCMOS) -- 3.4.11 Features of CCD and CMOS Cameras -- 3.4.12 Choosing a Digital Camera for Fluorescence Microscopy -- 3.4.13 Photomultiplier Tube (PMT) -- 3.4.14 Avalanche Photodiode (APD) -- 3.5 Types of Noise in a Digital Microscopy Image -- 3.6 Quantitative Fluorescence Microscopy -- 3.6.1 Measurements of Fluorescence Intensity and Concentration of the Labeled Target -- 3.6.2 Ratiometric Measurements (Ca++, pH) -- 3.6.3 Measurements of Dimensions in 3D Fluorescence Microscopy -- 3.6.4 Measurements of Exciting Light Intensity -- 3.6.5 Technical Tips for Quantitative Fluorescence Microscopy -- 3.7 Limitations of Fluorescence Microscopy -- 3.7.1 Photobleaching -- 3.7.2 Reversible Photobleaching under Oxidizing or Reducing Conditions -- 3.7.3 Phototoxicity -- 3.7.4 Optical ResolutionSummary: 3.7.5 Misrepresentation of Small Objects -- 3.8 Summary and Outlook -- References -- Chapter 4 Fluorescence Labeling -- 4.1 Introduction -- 4.2 Key Properties of Fluorescent Labels -- 4.3 Synthetic Fluorophores -- 4.3.1 Organic Dyes -- 4.3.2 Fluorescent Nanoparticles -- 4.3.3 Conjugation Strategies for Synthetic Fluorophores -- 4.3.4 Non-natural Amino Acids -- 4.3.5 Bringing the Fluorophore to Its Target -- 4.4 Genetically Encoded Labels -- 4.4.1 Phycobiliproteins -- 4.4.2 GFP-Like Proteins -- 4.5 Label Selection for Particular Applications -- 4.5.1 FRET to Monitor Intramolecular Conformational Dynamics -- 4.5.2 Protein Expression in Cells -- 4.5.3 Fluorophores as Sensors Inside the Cell -- 4.5.4 Live-Cell Dynamics -- 4.5.5 Super-Resolution Imaging -- 4.6 Summary -- References -- Chapter 5 Confocal Microscopy -- 5.1 Evolution and Limits of Conventional Widefield Microscopy -- 5.2 Theory of Confocal Microscopy -- 5.2.1 Principle of Confocal Microscopy -- 5.2.2 Radial and Axial Resolution and the Impact of the Pinhole Size -- 5.2.3 Scanning Confocal Imaging -- 5.2.3.1 Stage Scanning -- 5.2.3.2 Laser Scanning -- 5.2.3.3 Spinning Disk Confocal Microscope -- 5.2.4 Confocal Deconvolution -- 5.3 Applications of Confocal Microscopy -- 5.3.1 Nonscanning Applications -- 5.3.1.1 Fluorescence Correlation Spectroscopy -- 5.3.1.2 Fluorescence Cross-Correlation Spectroscopy -- 5.3.1.3 Pulsed Interleaved Excitation -- 5.3.1.4 Burst Analysis with Multiparameter Fluorescence Detection -- 5.3.2 Scanning Applications beyond Imaging -- 5.3.2.1 Number and Brightness Analysis -- 5.3.2.2 Raster Image Correlation Spectroscopy -- Acknowledgments -- References -- Chapter 6 Two-Photon Excitation Microscopy for Three-Dimensional Imaging of Living Intact Tissues -- 6.1 Introduction -- 6.2 What is Two-Photon Excitation? -- 6.2.1 Nonlinear Optics and 2PMSummary: 6.2.2 History and Theory of 2PM -- 6.3 How Does Two-Photon Excitation Microscopy Work in Practice? -- 6.3.1 The Role of Light Absorption in 2PM -- 6.3.2 The Role of Light Scattering in 2PM -- 6.4 Instrumentation -- 6.4.1 Lasers for 2PM -- 6.4.2 Detection Strategies for 2PM -- 6.4.3 The Advantages of 2PM for Deep-Tissue Imaging -- 6.5 Limitations of Two-Photon Excitation Microscopy -- 6.5.1 Limits of Spatial Resolution in 2PM -- 6.5.2 Potential Sample Heating by the High Laser Powers in 2PM -- 6.5.3 Difficulties in Predicting and Measuring Two-Photon Excitation Spectra -- 6.5.4 Accelerated Photobleaching (and Associated Photodamage) in the Focal Plane -- 6.5.5 Expensive Lasers Create a Practical Limitation for Some Experiments -- 6.6 When is 2PM the Best Option? -- 6.6.1 Thick Specimen including In Vivo Imaging -- 6.6.2 Imaging Fluorophores with Excitation Peaks in the Ultraviolet (UV) Spectrum -- 6.6.3 Localized Photochemistry -- 6.7 Applications of Two-Photon Microscopy -- 6.7.1 Imaging UV-Excited Fluorophores, such as NADH for Metabolic Activity -- 6.7.2 Localized Photoactivation of "Caged" Compounds -- 6.7.3 Imaging Electrical Activity in Deep Tissue -- 6.7.4 Light Sheet Microscopy Using Two-Photon Excitation -- 6.7.5 Other Applications of 2PM -- 6.8 Other Nonlinear Microscopies -- 6.9 Future Outlook for 2PM -- 6.10 Summary -- Acknowledgment -- References -- Chapter 7 Light Sheet Microscopy -- 7.1 Principle of Light Sheet Microscopy -- 7.2 Light Sheet Microscopy: Key Advantages -- 7.3 Construction and Working of a Light Sheet Microscope -- 7.4 Theory of Light Sheet Microscopy -- 7.5 Light Sheet Interaction with Tissue -- 7.6 3D Imaging -- 7.7 Multiview Imaging -- 7.8 Different Lens Configurations -- 7.9 Sample Mounting -- 7.10 Recent Advances in Light Sheet Microscopy -- 7.11 Outlook -- 7.11.1 Big DataSummary: 7.11.2 Smart Microscope: Imaging Concept of the Future -- 7.11.3 High-Throughput Imaging -- 7.12 Summary -- References -- Chapter 8 Localization-Based Super-Resolution Microscopy -- 8.1 Super-Resolution Microscopy: An Introduction -- 8.2 The Principle of Single-Molecule Localization Microscopy -- 8.3 Photoactivatable and Photoconvertible Probes -- 8.4 Intrinsically Photoswitchable Probes -- 8.5 Photoswitching of Organic Fluorophores by Chemical Reactions -- 8.6 Experimental Setup for Localization Microscopy -- 8.7 Optical Resolution and Imaging Artifacts -- 8.8 Fluorescence Labeling for Super-Resolution Microscopy -- 8.8.1 Label Size versus Structural Resolution -- 8.8.2 Live-Cell Labeling -- 8.8.3 Click Chemistry -- 8.8.4 Three-Dimensional SMLM -- 8.8.5 Astigmatic Imaging -- 8.8.6 Biplane Imaging -- 8.8.7 Double Helix PSF -- 8.8.8 Interferometric Imaging -- 8.9 Measures for Improving Imaging Contrast -- 8.10 SMLM Software -- 8.11 Reference Structures for SMLM -- 8.12 Quantification of SMLM Data -- 8.13 Summary -- References -- Chapter 9 Super-Resolution Microscopy: Interference and Pattern Techniques -- 9.1 Introduction -- 9.1.1 Review: The Resolution Limit -- 9.2 Structured Illumination Microscopy (SIM) -- 9.2.1 Image Generation in Structured Illumination Microscopy -- 9.2.2 Extracting the High-Resolution Information -- 9.2.3 Optical Sectioning by SIM -- 9.2.4 How the Illumination Pattern is Generated? -- 9.2.5 Mathematical Derivation of the Interference Pattern -- 9.2.6 Examples for SIM Setups -- 9.3 Spatially Modulated Illumination (SMI) Microscopy -- 9.3.1 Overview -- 9.3.2 SMI Setup -- 9.3.3 Excitation Light Distribution -- 9.3.4 Object Size Estimation with SMI Microscopy -- 9.4 Application of Patterned Techniques -- 9.5 Conclusion -- 9.6 Summary -- Acknowledgments -- References -- Chapter 10 STED Microscopy -- 10.1 IntroductionSummary: 10.2 The Concepts behind STED Microscopy
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Cover -- Title Page -- Copyright -- Contents -- List of Contributors -- Preface -- Chapter 1 Introduction to Optics -- 1.1 A Short History of Theories about Light -- 1.2 Properties of Light Waves -- 1.2.1 An Experiment on Interference -- 1.2.2 Physical Description of Light Waves -- 1.3 Four Effects of Interference -- 1.3.1 Diffraction -- 1.3.2 The Refractive Index -- 1.3.3 Refraction -- 1.3.4 Reflection -- 1.3.5 Light Waves and Light Rays -- 1.4 Optical Elements -- 1.4.1 Lenses -- 1.4.2 Metallic Mirrors -- 1.4.3 Dielectric Mirrors -- 1.4.4 Filters -- 1.4.5 Chromatic Reflectors -- 1.5 Optical Aberrations -- References -- Chapter 2 Principles of Light Microscopy -- 2.1 Introduction -- 2.2 Construction of Light Microscopes -- 2.2.1 Components of Light Microscopes -- 2.2.2 Imaging Path -- 2.2.3 Magnification -- 2.2.4 Angular and Numerical Aperture -- 2.2.5 Field of View -- 2.2.6 Illumination Beam Path -- 2.2.6.1 Critical and Köhler Illumination -- 2.2.6.2 Bright-Field and Epi-Illumination -- 2.3 Wave Optics and Resolution -- 2.3.1 Wave Optical Description of the Imaging Process -- 2.3.2 The Airy Pattern -- 2.3.3 Point Spread Function and Optical Transfer Function -- 2.3.4 Lateral and Axial Resolution -- 2.3.4.1 Lateral Resolution Using Incoherent Light Sources -- 2.3.4.2 Lateral Resolution of Coherent Light Sources -- 2.3.4.3 Axial Resolution -- 2.3.5 Magnification and Resolution -- 2.3.6 Depth of Field and Depth of Focus -- 2.3.7 Over- and Undersampling -- 2.4 Apertures, Pupils, and Telecentricity -- 2.5 Microscope Objectives -- 2.5.1 Objective Lens Design -- 2.5.2 Light Collection Efficiency and Image Brightness -- 2.5.3 Objective Lens Classes -- 2.5.4 Immersion Media -- 2.5.5 Special Applications -- 2.6 Contrast -- 2.6.1 Dark Field -- 2.6.2 Phase Contrast -- 2.6.2.1 Frits Zernike's Experiments -- 2.6.2.2 Setup of a Phase-Contrast Microscope

2.6.2.3 Properties of Phase-Contrast Images -- 2.6.3 Interference Contrast -- 2.6.4 Advanced Topic: Differential Interference Contrast -- 2.6.4.1 Optical Setup of a DIC Microscope -- 2.6.4.2 Interpretation of DIC Images -- 2.6.4.3 Comparison between DIC and Phase Contrast -- 2.7 Summary -- Acknowledgments -- References -- Chapter 3 Fluorescence Microscopy -- 3.1 Contrast in Optical Microscopy -- 3.2 Physical Foundations of Fluorescence -- 3.2.1 What is Fluorescence? -- 3.2.2 Fluorescence Excitation and Emission Spectra -- 3.3 Features of Fluorescence Microscopy -- 3.3.1 Image Contrast -- 3.3.2 Specificity of Fluorescence Labeling -- 3.3.3 Sensitivity of Detection -- 3.4 A Fluorescence Microscope -- 3.4.1 Principle of Operation -- 3.4.2 Sources of Exciting Light -- 3.4.3 Optical Filters in a Fluorescence Microscope -- 3.4.4 Electronic Filters -- 3.4.5 Photodetectors for Fluorescence Microscopy -- 3.4.6 CCD or Charge-Coupled Device -- 3.4.7 Intensified CCD (ICCD) -- 3.4.8 Electron-Multiplying Charge-Coupled Device (EMCCD) -- 3.4.9 CMOS -- 3.4.10 Scientific CMOS (sCMOS) -- 3.4.11 Features of CCD and CMOS Cameras -- 3.4.12 Choosing a Digital Camera for Fluorescence Microscopy -- 3.4.13 Photomultiplier Tube (PMT) -- 3.4.14 Avalanche Photodiode (APD) -- 3.5 Types of Noise in a Digital Microscopy Image -- 3.6 Quantitative Fluorescence Microscopy -- 3.6.1 Measurements of Fluorescence Intensity and Concentration of the Labeled Target -- 3.6.2 Ratiometric Measurements (Ca++, pH) -- 3.6.3 Measurements of Dimensions in 3D Fluorescence Microscopy -- 3.6.4 Measurements of Exciting Light Intensity -- 3.6.5 Technical Tips for Quantitative Fluorescence Microscopy -- 3.7 Limitations of Fluorescence Microscopy -- 3.7.1 Photobleaching -- 3.7.2 Reversible Photobleaching under Oxidizing or Reducing Conditions -- 3.7.3 Phototoxicity -- 3.7.4 Optical Resolution

3.7.5 Misrepresentation of Small Objects -- 3.8 Summary and Outlook -- References -- Chapter 4 Fluorescence Labeling -- 4.1 Introduction -- 4.2 Key Properties of Fluorescent Labels -- 4.3 Synthetic Fluorophores -- 4.3.1 Organic Dyes -- 4.3.2 Fluorescent Nanoparticles -- 4.3.3 Conjugation Strategies for Synthetic Fluorophores -- 4.3.4 Non-natural Amino Acids -- 4.3.5 Bringing the Fluorophore to Its Target -- 4.4 Genetically Encoded Labels -- 4.4.1 Phycobiliproteins -- 4.4.2 GFP-Like Proteins -- 4.5 Label Selection for Particular Applications -- 4.5.1 FRET to Monitor Intramolecular Conformational Dynamics -- 4.5.2 Protein Expression in Cells -- 4.5.3 Fluorophores as Sensors Inside the Cell -- 4.5.4 Live-Cell Dynamics -- 4.5.5 Super-Resolution Imaging -- 4.6 Summary -- References -- Chapter 5 Confocal Microscopy -- 5.1 Evolution and Limits of Conventional Widefield Microscopy -- 5.2 Theory of Confocal Microscopy -- 5.2.1 Principle of Confocal Microscopy -- 5.2.2 Radial and Axial Resolution and the Impact of the Pinhole Size -- 5.2.3 Scanning Confocal Imaging -- 5.2.3.1 Stage Scanning -- 5.2.3.2 Laser Scanning -- 5.2.3.3 Spinning Disk Confocal Microscope -- 5.2.4 Confocal Deconvolution -- 5.3 Applications of Confocal Microscopy -- 5.3.1 Nonscanning Applications -- 5.3.1.1 Fluorescence Correlation Spectroscopy -- 5.3.1.2 Fluorescence Cross-Correlation Spectroscopy -- 5.3.1.3 Pulsed Interleaved Excitation -- 5.3.1.4 Burst Analysis with Multiparameter Fluorescence Detection -- 5.3.2 Scanning Applications beyond Imaging -- 5.3.2.1 Number and Brightness Analysis -- 5.3.2.2 Raster Image Correlation Spectroscopy -- Acknowledgments -- References -- Chapter 6 Two-Photon Excitation Microscopy for Three-Dimensional Imaging of Living Intact Tissues -- 6.1 Introduction -- 6.2 What is Two-Photon Excitation? -- 6.2.1 Nonlinear Optics and 2PM

6.2.2 History and Theory of 2PM -- 6.3 How Does Two-Photon Excitation Microscopy Work in Practice? -- 6.3.1 The Role of Light Absorption in 2PM -- 6.3.2 The Role of Light Scattering in 2PM -- 6.4 Instrumentation -- 6.4.1 Lasers for 2PM -- 6.4.2 Detection Strategies for 2PM -- 6.4.3 The Advantages of 2PM for Deep-Tissue Imaging -- 6.5 Limitations of Two-Photon Excitation Microscopy -- 6.5.1 Limits of Spatial Resolution in 2PM -- 6.5.2 Potential Sample Heating by the High Laser Powers in 2PM -- 6.5.3 Difficulties in Predicting and Measuring Two-Photon Excitation Spectra -- 6.5.4 Accelerated Photobleaching (and Associated Photodamage) in the Focal Plane -- 6.5.5 Expensive Lasers Create a Practical Limitation for Some Experiments -- 6.6 When is 2PM the Best Option? -- 6.6.1 Thick Specimen including In Vivo Imaging -- 6.6.2 Imaging Fluorophores with Excitation Peaks in the Ultraviolet (UV) Spectrum -- 6.6.3 Localized Photochemistry -- 6.7 Applications of Two-Photon Microscopy -- 6.7.1 Imaging UV-Excited Fluorophores, such as NADH for Metabolic Activity -- 6.7.2 Localized Photoactivation of "Caged" Compounds -- 6.7.3 Imaging Electrical Activity in Deep Tissue -- 6.7.4 Light Sheet Microscopy Using Two-Photon Excitation -- 6.7.5 Other Applications of 2PM -- 6.8 Other Nonlinear Microscopies -- 6.9 Future Outlook for 2PM -- 6.10 Summary -- Acknowledgment -- References -- Chapter 7 Light Sheet Microscopy -- 7.1 Principle of Light Sheet Microscopy -- 7.2 Light Sheet Microscopy: Key Advantages -- 7.3 Construction and Working of a Light Sheet Microscope -- 7.4 Theory of Light Sheet Microscopy -- 7.5 Light Sheet Interaction with Tissue -- 7.6 3D Imaging -- 7.7 Multiview Imaging -- 7.8 Different Lens Configurations -- 7.9 Sample Mounting -- 7.10 Recent Advances in Light Sheet Microscopy -- 7.11 Outlook -- 7.11.1 Big Data

7.11.2 Smart Microscope: Imaging Concept of the Future -- 7.11.3 High-Throughput Imaging -- 7.12 Summary -- References -- Chapter 8 Localization-Based Super-Resolution Microscopy -- 8.1 Super-Resolution Microscopy: An Introduction -- 8.2 The Principle of Single-Molecule Localization Microscopy -- 8.3 Photoactivatable and Photoconvertible Probes -- 8.4 Intrinsically Photoswitchable Probes -- 8.5 Photoswitching of Organic Fluorophores by Chemical Reactions -- 8.6 Experimental Setup for Localization Microscopy -- 8.7 Optical Resolution and Imaging Artifacts -- 8.8 Fluorescence Labeling for Super-Resolution Microscopy -- 8.8.1 Label Size versus Structural Resolution -- 8.8.2 Live-Cell Labeling -- 8.8.3 Click Chemistry -- 8.8.4 Three-Dimensional SMLM -- 8.8.5 Astigmatic Imaging -- 8.8.6 Biplane Imaging -- 8.8.7 Double Helix PSF -- 8.8.8 Interferometric Imaging -- 8.9 Measures for Improving Imaging Contrast -- 8.10 SMLM Software -- 8.11 Reference Structures for SMLM -- 8.12 Quantification of SMLM Data -- 8.13 Summary -- References -- Chapter 9 Super-Resolution Microscopy: Interference and Pattern Techniques -- 9.1 Introduction -- 9.1.1 Review: The Resolution Limit -- 9.2 Structured Illumination Microscopy (SIM) -- 9.2.1 Image Generation in Structured Illumination Microscopy -- 9.2.2 Extracting the High-Resolution Information -- 9.2.3 Optical Sectioning by SIM -- 9.2.4 How the Illumination Pattern is Generated? -- 9.2.5 Mathematical Derivation of the Interference Pattern -- 9.2.6 Examples for SIM Setups -- 9.3 Spatially Modulated Illumination (SMI) Microscopy -- 9.3.1 Overview -- 9.3.2 SMI Setup -- 9.3.3 Excitation Light Distribution -- 9.3.4 Object Size Estimation with SMI Microscopy -- 9.4 Application of Patterned Techniques -- 9.5 Conclusion -- 9.6 Summary -- Acknowledgments -- References -- Chapter 10 STED Microscopy -- 10.1 Introduction

10.2 The Concepts behind STED Microscopy

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