Title page -- Copyright page -- Contents -- Preface -- Acknowledgments -- CHAPTER 1: Fundamentals of Light Microscopy -- Overview -- Optical Components of the Light Microscope -- Aperture and Image Planes in a Focused, Adjusted Microscope -- Koehler Illumination -- Adjusting the Microscope for Koehler Illumination -- Fixed Tube Length versus Infinity Optical Systems -- Precautions for Handling Optical Equipment -- Care and Maintenance of the Microscope -- CHAPTER 2: Light and Color -- Overview -- Light as a Probe of Matter -- The Dual Particle- and Wave-Like Nature of Light -- The Quality of Light -- Properties of Light Perceived by the Eye -- Physical Basis for Visual Perception and Color -- Addition and Subtraction Colors -- CHAPTER 3: Illuminators, Filters, and the Isolation of Specific Wavelengths -- Overview -- Illuminators and Their Spectra -- Illuminator Alignment and Bulb Replacement -- Filters for Adjusting the Intensity and Wavelength of Illumination -- Neutral Density Filters -- Colored Glass Filters -- Interference Filters -- Effects of Light on Living Cells -- CHAPTER 4: Lenses and Geometrical Optics -- Overview -- Reflection and Refraction of Light -- Image Formation by a Simple Lens -- Rules of Ray Tracing for a Simple Lens -- Object-Image Math -- The Principal Aberrations of Lenses -- Designs and Specifications of Objectives -- Special Objective Designs -- Markings on the Barrel of an Objective -- Image Brightness -- Objective Parfocality -- Condensers -- Oculars -- Microscope Slides and Coverslips -- The Care and Cleaning of Optics -- Dust -- Immersion Oil -- Scratches and Abrasions -- Mechanical Force -- CHAPTER 5: Diffraction and Interference in Image Formation -- Overview -- Diffraction and Interference -- The Diffraction Image of a Point Source of Light -- The Constancy of Optical Path Length between Object and Image.

Effect of Aperture Angle on Diffraction Spot Size -- Diffraction by a Grating and Calculation of Its Line Spacing, D -- Abbé's Theory for Image Formation in the Microscope -- A Diffraction Pattern Is Formed in the Rear Aperture of the Objective -- Preservation of Coherence: Essential Requirement for Image Formation -- CHAPTER 6: Diffraction and Spatial Resolution -- Overview -- Numerical Aperture -- Spatial Resolution -- Depth of Field and Depth of Focus -- Optimizing the Microscope Image: A Compromise between Spatial Resolution and Contrast -- CHAPTER 7: Phase Contrast Microscopy and Darkfield Microscopy -- Overview -- Phase Contrast Microscopy -- The Behavior of Waves from Phase Objects in Brightfield Microscopy -- Wave Terminology and the Importance of Coherence -- Depiction of Wave Interactions with Sine Wave and Vector Diagrams -- The Role of Differences in Optical Path Lengths -- The Optical Design of the Phase Contrast Microscope -- Alignment -- Interpreting the Phase Contrast Image -- Darkfield Microscopy -- Theory and Optics -- Image Interpretation -- CHAPTER 8: Properties of Polarized Light -- Overview -- The Generation of Polarized Light -- Polarization by Reflection and Scattering -- Vectorial Analysis of Polarized Light Using a Dichroic Filter -- Double Refraction in Crystals -- Kinds of Birefringence -- Propagation of O and E Wavefronts in a Birefringent Crystal -- Birefringence in Biological Specimens -- Generation of Elliptically Polarized Light by Birefringent Specimens -- CHAPTER 9: Polarization Microscopy -- Overview -- Optics of the Polarizing Microscope -- Adjusting the Polarizing Microscope -- Appearance of Birefringent Objects in Polarized Light -- Principles of Action of Retardation Plates and Three Popular Compensators -- λ-Plate Compensator -- de Sénarmont Compensator -- Brace-Koehler Compensator.

CHAPTER 10: Differential Interference Contrast Microscopy and Modulation Contrast Microscopy -- Overview -- The DIC Optical System -- DIC Equipment and Optics -- The DIC Prism -- Formation of the DIC Image -- Interference between O and E Wavefronts and the Application of Bias Retardation -- Alignment of DIC Components -- Image Interpretation -- The Use of Compensators in DIC Microscopy -- Comparison of DIC and Phase Contrast Optics -- Modulation Contrast Microscopy -- Contrast Methods Using Oblique Illumination -- Alignment of the Modulation Contrast Microscope -- CHAPTER 11: Fluorescence Microscopy -- Overview -- Applications of Fluorescence Microscopy -- Physical Basis of Fluorescence -- Properties of Fluorescent Dyes -- Autofluorescence of Endogenous Molecules -- Fluorescent Dyes and Proteins in Fluorescence Microscopy -- Alexa Fluor Dyes -- Cyanine Dyes -- Fluorescent Environmental Probes -- Organelle Probes -- Quantum Dots -- Fluorescent Proteins -- Arrangement of Filters and the Epi-Illuminator in the Fluorescence Microscope -- Lamps for Fluorescence Excitation -- The Epi-Illuminator -- Filters -- The Dichromatic Mirror -- Objectives and Spatial Resolution in Fluorescence Microscopy -- Causes of High Fluorescence Background -- The Problem of Bleedthrough with Multiply Stained Specimens -- Quenching, Blinking, and Photobleaching -- Examining Fluorescent Molecules in Living Cells -- CHAPTER 12: Fluorescence Imaging of Dynamic Molecular Processes -- Overview -- Modes of Dynamic Fluorescence Imaging -- Förster Resonance Energy Transfer -- The Molecular Mechanism of FRET -- FRET Probes -- Paradigms for FRET -- Evaluating FRET from Fluorescence Images -- Methods for Detecting FRET -- Applications -- Fluorescence Recovery after Photobleaching -- A Molecular Model for Analyzing FRAP Measurements -- Measurement of Diffusion Coefficients Using FRAP.

FRAP Examples -- FRAP Equipment -- FRAP Controls -- TIRF Microscopy: Excitation by an Evanescent Wave -- Total Internal Reflection -- The Evanescent Wave -- Microscope Configurations Used to Obtain TIRF -- TIRF Objectives -- The TIRF Illuminator -- Filters for TIRF -- Advanced and Emerging Dynamic Fluoresence Techniques -- Fluorescence Lifetime Imaging Microscopy -- Fluorescence Correlation Spectroscopy -- Harmonic Generation Microscopy -- Coherent Anti-Stokes Raman Scattering Microscopy -- Fluorescence Speckle Microscopy -- CHAPTER 13: Confocal Laser Scanning Microscopy -- Overview -- The Optical Principle of Confocal Imaging -- Advantages of CLSM over Widefield Fluorescence Systems -- Criteria Defining Image Quality and the Performance of an Electronic Imaging System -- Confocal Adjustments and Their Effects on Imaging -- Pinhole Adjustment -- Scan Rate, Image Size, and Dwell Time -- Zoom Factor -- Lasers for Fluorophore Excitation -- Gain and Offset Settings of the PMT Detector -- Objective Selection -- Photobleaching -- General Procedure for Acquiring a Confocal Image -- Performance Check of a Confocal System -- Fast (Real-Time) Imaging in Confocal Microscopy -- Confocal Imaging with a Spinning Disk -- Sweptfield Confocal Imaging -- Resonant Scanning Confocal Microscopy -- Additional Options for Rapid Confocal Imaging -- Spectral Analysis: A Valuable Enhancement for Confocal Imaging -- Optical Sectioning by Structured Illumination -- Deconvolution Microscopy -- Microscope Configuration for Deconvolution -- Determination of the Point-Spread Function -- Deconvolution Algorithms -- Evaluating Deconvolution Results -- CHAPTER 14: Two-Photon Excitation Fluorescence Microscopy -- Overview -- The Problem of Photon Scattering in Deep Tissue Imaging -- Two-Photon Excitation Is a Nonlinear Process -- Localization of Excitation.

Why Two-Photon Imaging Works -- Resolution -- Equipment -- Ti:Sapphire Laser -- Scan Head -- Detectors -- Examples and Applications of Two-Photon Fluorescence Microscopy -- Three-Photon Excitation -- Second Harmonic Generation Microscopy -- CHAPTER 15: Superresolution Imaging -- Overview -- The RESOLFT Concept -- Single-Molecule Localization Microscopy -- Special Requirements and Conditions for PALM/STORM Imaging -- Microscope Configuration for Single-Molecule Superresolution -- Modes of Single-Molecule Localization Microscopy -- Fluorescent Probes for PALM/STORM Imaging -- Structured Illumination Microscopy -- NA Limits the Spatial Resolution in an Image -- High Frequency Information Is Contained in Low Frequency Moiré Fringes -- Spatial Frequencies Are Extracted from SIM Images, Extending Resolution Twofold -- Important Guidelines and Conditions for SIM Microscope Optics -- SIM for 3D, Multicolor Imaging of Living Cells in Time-Lapse Mode -- Stimulated Emission Depletion (STED) Microscopy: Superresolution by PSF Engineering -- Shaping the Scanning Spot -- Shaping the PSF by Ground-State-Depletion (GSD)-An Alternative to STED -- CHAPTER 16: Imaging Living Cells with the Microscope -- Overview -- Labeling Strategies for Live-Cell Imaging -- Introducing Fluorescent Proteins into Living Cells -- Labeling with Synthetic Fluorophores -- Hybrid Labeling Systems -- Control of Illumination -- Phototoxicity and Photodamage -- Monitoring Cell Viability and Variability -- Control of Environmental Conditions -- Keeping Cells Alive and Healthy on the Microscope Stage -- Live-Cell Imaging Chambers -- Laboratory Environment and Isolation of Vibration -- Optics, Detectors, and Hardware -- Focus Drift -- Optimizing Signal-to-Noise with Binning, Gain, and Exposure Time -- Microscope Optics for Live-Cell Imaging -- Time-Lapse Imaging -- Incubator Microscopes.

Evaluating Live-Cell Imaging Results.