A Practical Guide to Optical Microscopy.
- 1st ed.
- 1 online resource (279 pages)
Cover -- Half Title -- Title Page -- Copyright Page -- Contents -- Preface -- Acknowledgements -- About the Author -- 1: Introduction -- 1.1 A Historical Perspective -- 1.2 Initial Considerations on which Method to Choose for a Specific Application -- 1.3 How to Use This Book -- References -- 2: Understanding Light in Optical Microscopy -- 2.1 Introduction to the Physics of Optical Microscopy -- 2.1.1 Light -- 2.1.2 Waves, Wavelength, Frequency and Particles -- 2.1.3 Refractive Index -- 2.1.4 Polarization -- 2.2 The Optics of Microscopy -- 2.2.1 Refraction -- 2.2.2 Interference and Diffraction -- 2.3 Limitations in Optical Microscopy -- 2.3.1 Chromatic Aberration -- 2.3.2 Spherical Aberration -- 2.3.3 Astigmatism -- 2.3.4 Field Curvature -- 2.4 Contrast Mechanisms -- 2.4.1 Absorption -- 2.4.2 Scattering -- 2.4.3 Phase or Refractive Index Changes -- 2.4.4 Fluorescence -- 2.4.5 Fluorescence Lifetime -- 2.4.6 Non-linear Excitation -- Harmonic Generation, Raman Scattering -- 2.5 A Brief Introduction to Light Sources for Microscopy -- 2.5.1 Conventional Filament Bulb (Including Metal Halide) -- 2.5.2 Mercury Vapour Bulb -- 2.5.3 Light Emitting Diodes (LEDs) -- 2.5.4 Arc Lamp -- 2.5.5 Lasers -- 2.6 Detection of Light in Microscopy -- 2.6.1 Human Eye and Photographic Film -- 2.6.2 Electronic or Digital Camera -- 2.6.3 Photomultiplier -- 2.6.4 Photodiodes -- References -- 3: Basic Microscope Optics -- 3.1 Introduction -- 3.2 Basic Types of Widefield Optical Microscope -- 3.3 Core Optics of a Widefield Microscope -- 3.4 Optimal Illumination -- 3.5 Microscope Objectives -- 3.6 Image Detection and Recording -- 3.7 Guidelines for Use and Advantages and Disadvantages of a Widefield Microscope -- 4: Advanced Widefield Microscopy -- 4.1 Introduction -- 4.2 Polarization Microscopy -- 4.2.1 Practical Implementation of Polarization Microscopy. 4.2.2 Applications of Polarization Microscopy -- 4.3 Phase Contrast Microscopy -- 4.3.1 Practical Implementation of Phase Contrast Microscopy -- 4.3.2 Applications of Phase Microscopy -- 4.4 Differential Interference Contrast (DIC) Microscopy -- 4.4.1 Practical Implementation of DIC Microscopy -- 4.4.2 Applications of DIC Microscopy -- 4.5 Darkfield Microscopy -- 4.5.1 Practical Implementation of Darkfield Microscopy -- 4.5.2 Practical Applications of Darkfield Microscopy -- 4.6 Fluorescence Microscopy -- 4.6.1 Practical Implementation of Fluorescence Microscopy -- 4.6.2 Practical Applications of Fluorescence Microscopy -- 4.7 Summary and Method Selection -- 5: Confocal Microscopy -- 5.1 Principles of Confocal Microscopy -- 5.2 Beam Scanned Confocal System -- 5.3 Filter Selection for Beam Scanned Confocal Systems -- 5.4 Detector Selection for Beam Scanned Confocal Systems -- 5.5 Nipkow or Spinning Disk Confocal Systems -- 5.6 Practical Guidelines to Maximize the Performance of a Confocal Microscope -- 5.6.1 Microscope Choice, Sample Mounting and Preparation -- 5.6.2 Lens Selection -- 5.6.3 Initial Image Capture -- 5.6.4 Optimization -- 5.6.5 Saving Data -- 5.6.6 Routine Maintenance -- 5.7 Reconstruction -- References -- 6: Fluorescence Lifetime Imaging Microscopy (FLIM) -- 6.1 Introduction to Fluorescence Lifetime -- 6.1.1 Absorption and Emission -- 6.1.2 Fluorescence Lifetime -- 6.2 Measurement Techniques -- 6.2.1 Time Correlated Single Photon Counting (TCSPC) -- 6.2.2 Time Gating Electronics -- 6.2.3 Time Gated Camera -- 6.2.4 Phase Measurement -- 6.2.5 Slow Detector Method -- 6.3 Methods of Analysis -- 6.4 Experimental Considerations and Guidelines for Use -- 6.4.1 FLIM for Enhancing Contrast -- 6.4.2 FLIM for FRET and Observing Changes in Lifetime -- 6.4.3 FLIM for Absolute Lifetime Measurement -- 6.4.4 Practical Considerations -- References. 7: Light Sheet or Selective Plane Microscopy -- 7.1 Introduction -- 7.2 Brief History of Light Sheet Microscopy -- 7.3 Optical Principles -- 7.4 Practical Systems -- 7.4.1 Optical Details -- 7.4.2 Basic Alignment -- 7.4.3 Basic Variations in SPIM -- 7.5 Practical Operation -- 7.5.1 Sample Mounting -- 7.5.2 Basic Operation -- 7.5.3 Correcting Common Faults -- 7.5.4 High Speed Imaging and Synchronization -- 7.5.5 High Throughput SPIM -- 7.6 SPIM Imaging Processing -- 7.7 Advanced SPIM Methods -- 7.8 What Is SPIM Good for and Limitations -- References -- 8: Multiphoton Fluorescence Microscopy -- 8.1 Introduction to Multiphoton Excitation -- 8.1.1 Requirements on Light Sources for Multiphoton Excitation -- 8.1.2 Requirements on Photon Detection for Multiphoton Microscopy -- 8.2 Practical Multiphoton Microscopy -- 8.2.1 Wavelength -- 8.2.2 Pulse Width and Dispersion Compensation -- 8.2.3 Average Power -- 8.2.4 Objective Lens Selection -- 8.2.5 Detection -- 8.3 Going from Confocal to Multiphoton Microscopy -- 8.4 Advanced Multiphoton Microscopy -- 8.4.1 Endoscopic Multiphoton Microscopy -- 8.4.2 Adaptive Optics for Aberration Correction -- 8.4.3 Measurement of Two Photon "Dose" -- References -- 9: Harmonic Microscopy -- 9.1 Physical Basis for Harmonic Generation -- 9.2 Practical Harmonic Microscopy -- 9.3 Applications of Harmonic Microscopy -- References -- 10: Raman Microscopy -- 10.1 Physical Basis of the Raman Effect -- 10.2 Coherent Anti-Stokes Raman Scattering (CARS) -- 10.3 Stimulated Raman Scattering (SRS) Microscopy -- 10.4 Practical Raman Microscopy Instrumentation -- 10.5 Practical CARS Microscopy Instrumentation -- 10.6 Techniques and Applications in Raman Microscopy -- 10.7 Techniques and Applications in CARS and SRS Microscopy -- 10.8 When to Consider the Use of Raman Microscopy -- References -- 11: Digital Holographic Microscopy. 11.1 Physical Basis of the Method -- 11.2 Practical Implementation -- 11.2.1 The Light Source for Holographic Microscopy -- 11.2.2 The Detector for Holographic Microscopy -- 11.2.3 The Reconstruction Algorithm for Holographic Microscopy -- 11.3 Practical Applications of Digital Holographic Microscopy -- 11.3.1 Surface Microscopy -- 11.3.2 Particle Tracking -- 11.3.3 Cell Imaging -- 11.3.4 Total Internal Reflection Digital Holographic Microscopy -- 11.4 Summary -- References -- 12: Super Resolution Microscopy -- 12.1 Introduction -- 12.2 Total Internal Reflection Microscopy -- 12.2.1 Principles of Total Internal Reflection Microscopy -- 12.2.2 Practical Implementations of Total Internal Reflection Microscopy -- 12.2.3 Practical Considerations for Total Internal Reflection Microscopy -- 12.3 Structured Illumination Microscopy (SIM) -- 12.3.1 Principles of Structured Illumination Microscopy -- 12.3.2 Practical Implementations of Structured Illumination Microscopy -- 12.3.3 Practical Considerations for Structured Illumination Microscopy -- 12.4 Localization Microscopy (STORM/PALM) -- 12.4.1 Principles of Localization Microscopy -- 12.4.2 Practical Implementations of Localization Microscopy -- 12.4.3 Practical Considerations for Localization Microscopy -- 12.5 Stimulated Emission and Depletion (STED) Microscopy -- 12.5.1 Principles of STED -- 12.5.2 Practical Implementations of STED -- 12.5.3 Practical Considerations for STED -- 12.6 Selection of Super-Resolution Methods -- References -- 13: How to Obtain the Most from Your Data -- 13.1 Introduction -- 13.2 Basics of Data Collection -- 13.3 Software Considerations -- 13.3.1 Open Source Image Processing Packages -- 13.3.2 Programming Languages for Image Processing -- 13.3.3 Commercial Image Processing Packages -- 13.4 Basics of Data Processing -- 13.4.1 Core Techniques -- 13.4.1.1 Reducing Noise. 13.4.1.2 Uneven Illumination -- 13.4.1.3 Increasing Contrast -- 13.4.1.4 Enhancing Perceived Detail -- 13.4.1.5 Monochrome Look-Up Tables and the Addition of Colour -- 13.5 Producing Quantified Data from Images -- 13.5.1 Intensity-Based Quantification -- 13.5.2 Spatial-Based Quantification -- 13.5.3 Temporal-Based Quantification -- 13.6 Deconvolution -- 13.7 Summary -- References -- 14: Selection Criteria for Optical Microscopy -- 14.1 Introduction -- 14.2 Basic Selection Guidelines -- 14.3 Specialized Techniques -- 14.3.1 Fluorescence Recovery after Photobleaching (FRAP) -- 14.3.2 Förster Resonant Energy Transfer (FRET) -- 14.3.3 Opto-genetics, Observation of Cell Ablation and Photo-uncaging -- 14.3.4 Imaging of Plants and Plant Cells -- 14.4 Summary -- References -- Glossary -- Index.
This book is aimed at providing users with a practical guide to help them select, and then use, the most suitable method for their application. It is an invaluable tool for use within research groups and laboratories in the life and physical sciences.