Introduction to optical microscopy

Description

This fully updated, self-contained textbook covering modern optical microscopy equips students with a solid understanding of the theory underlying a range of advanced techniques. Two new chapters cover pump-probe techniques, and imaging in scattering media, and additional material throughout covers light-sheet microscopy, image scanning microscopy, and much more. An array of practical techniques are discussed, from classical phase contrast and confocal microscopy, to holographic, structured illumination, multi-photon, and coherent Raman microscopy, and optical coherence tomography. Fundamental topics are also covered, including Fourier optics, partial coherence, 3D imaging theory, statistical optics, and the physics of scattering and fluorescence. With a wealth of end-of-chapter problems, and a solutions manual for instructors available online, this is an invaluable book for electrical engineering, biomedical engineering, and physics students taking graduate courses on optical microscopy, as well as advanced undergraduates, professionals, and researchers looking for an accessible introduction to the field. Contents Preface to Second Edition Preface to First Edition 1 Introduction 1.1 Complex Fields 1.2 Intensity and Radiance 1.3 Ray Optics 1.4 Basic Transfer Properties of a Lens 1.4.1 Fourier Transform with a Lens 1.4.2 Imaging with a Lens 1.4.3 Thick Lens 1.5 Problems References 2 Monochromatic Wave Propagation 2.1 Time-Dependent Wave Equation 2.2 Time-Independent Wave Equation 2.2.1 Propagating Versus Evanescent Waves 2.3 Rayleigh–Sommerfeld Diffraction 2.3.1 Primary Versus Secondary Sources 2.3.2 Propagator Properties 2.4 Approximations 2.4.1 Fresnel Diffraction 2.4.2 Fraunhofer Diffraction 2.5 Problems References 3 Monochromatic Field Propagation Through a Lens 3.1 Lens Transmission 3.2 Fourier Transform with a Lens 3.2.1 Gaussian Pupil 3.3 Imaging with a Lens 3.3.1 Arbitrary Pupil 3.3.2 4f Imaging 3.3.3 Amplitude Spread and Transfer Functions 3.3.4 Spatial Bandwidth and Resolution 3.3.5 Properties of Amplitude Spread Functions 3.4 Effect of Surrounding Media 3.5 Problems References 4 Intensity Propagation 4.1 Field Correlations 4.2 Free Space Propagation 4.2.1 Quasi-Homogeneous Beams 4.2.2 Reciprocity Between Coherence and Intensity 4.2.3 Incoherent Beam Propagation 4.3 Propagation Through a 2f System 4.4 Propagation Through a 4f System: Imaging 4.4.1 Circular Pupil 4.5 Problems References 5 3D Imaging 5.1 Defocus 5.1.1 Gaussian Beam 5.2 3D Imaging Functions 5.2.1 3D Coherent Transfer Function 5.2.2 3D Amplitude Spread Function 5.2.3 3D Point Spread Function 5.2.4 3D Optical Transfer Function 5.3 3D Imaging Examples 5.4 Frequency Support 5.4.1 Optical Sectioning 5.4.2 Extended Depth of Field 5.4.3 Radiant Mutual Intensity 5.5 Problems References 6 Radiometry 6.1 Energy Flux Density 6.1.1 Transport of Intensity 6.2 Radiance 6.2.1 Relation Between Flux Density and Radiance 6.3 Étendue 6.3.1 Conservation of Étendue 6.3.2 Multimode Beam Propagation 6.4 Throughput 6.4.1 Apparent Source Coherence 6.4.2 Transmission of Power 6.5 Microscope Layout 6.5.1 Field and Aperture Stops 6.5.2 Illumination Configurations 6.5.3 Detection Configuration 6.6 Problems References 7 Intensity Fluctuations 7.1 Temporal Coherence 7.1.1 Quasi-Stationary Beams 7.2 Coherence Functions 7.2.1 Interrupted Phase Model 7.2.2 Gaussian Light 7.2.3 Köhler Illumination 7.2.4 Central Limit Theorem 7.3 Filtered Fluctuations 7.3.1 Temporal Filtering 7.3.2 Spatial Integration 7.4 Coherence Examples 7.5 Problems References 8 Detection Noise 8.1 Shot Noise 8.1.1 Power Spectral Density 8.1.2 Gaussian Light Example 8.1.3 Unit Conversion 8.2 Other Noise Sources 8.2.1 Dark-Current Noise 8.2.2 Avalanche Noise 8.2.3 Johnson Noise 8.2.4 Noise Equivalent Power 8.2.5 Example: Split Detector 8.3 Cameras 8.3.1 Readout Noise 8.3.2 Photoelectron Preamplification 8.4 Electronic Gain Versus Optical Gain 8.5 Problems References 9 Absorption and Scattering 9.1 2D Formalism 9.1.1 Thin Sample Approximation 9.1.2 Plane-Wave Illumination 9.1.3 Beam Propagation Method 9.2 3D Formalism 9.2.1 Born Approximation 9.2.2 Rytov Approximation 9.3 Cross-Sections 9.3.1 Scattering Amplitude 9.3.2 Optical Theorem 9.4 Vector Dipole Scattering 9.5 Problems References 10 Widefield Microscopy 10.1 Transmission Microscopy 10.1.1 Thin Samples 10.1.2 Brightfield Microscopy 10.1.3 Phase Imaging 10.1.4 Volume Samples 10.2 Reflection Microscopy 10.2.1 Brightfield Microscopy 10.2.2 Darkfield Microscopy 10.3 Problems References 11 Interference Microscopy 11.1 Principle 11.2 Coherent Illumination 11.2.1 Fresnel Holography 11.2.2 Lensless Fourier Holography 11.2.3 Fourier Holography with a Lens 11.2.4 Imaging Holography 11.3 Incoherent Illumination 11.4 Frequency Support 11.4.1 Origin of Reference Beam 11.4.2 Extraction of Complex Interference 11.5 Resolution and Sampling 11.6 Applications 11.7 Problems References 12 Optical Coherence Tomography 12.1 Coherence Gating 12.2 Extraction of Complex Interference 12.2.1 Static Sample 12.2.2 Dynamic Sample 12.3 OCT Implementations 12.3.1 Time-Domain OCT 12.3.2 Frequency-Domain OCT 12.3.3 Comparison 12.4 Widefield OCT 12.4.1 Frequency Support 12.5 Problems References 13 Fluorescence 13.1 Rate Equations 13.1.1 Two-Level Fluorescent Molecule 13.1.2 Three-Level Molecule: Triplet State 13.1.3 Photobleaching 13.2 Förster Resonance Energy Transfer (FRET) 13.3 Fluorescence Lifetime Imaging Microscopy (FLIM) 13.3.1 Time Domain 13.3.2 Frequency Domain 13.4 Fluorescence Correlation Spectroscopy (FCS) 13.4.1 Molecular Diffusion 13.4.2 Example: Hard-Sphere Volume with Photobleaching 13.5 Problems References 14 Confocal Microscopy 14.1 Scanning Configurations 14.2 Non-Fluorescence Confocal Microscopy 14.2.1 Transmission Confocal 14.2.2 Reflection Confocal 14.2.3 Comparison with Widefield Microscopy 14.3 Confocal Fluorescence Microscopy 14.3.1 Optical Sectioning 14.3.2 Confocal Probe Volume 14.3.3 Frequency Support 14.3.4 Finite-Size Pinhole 14.3.5 Offset Illumination and Detection Pupils 14.4 Problems References 15 Structured Illumination Microscopy 15.1 Principles 15.2 Structured Illumination with Fringes 15.2.1 Optical Sectioning 15.2.2 Coherent Versus Incoherent Illumination 15.3 Structured Illumination with Speckle 15.3.1 Decoding with Known Patterns 15.3.2 Decoding with Unknown Patterns 15.4 Hybrid Techniques 15.4.1 Aperture Correlation Microscopy 15.4.2 Programmable Array Microscopy 15.5 Pupil Synthesis 15.6 Problems References 16 Multiphoton Microscopy 16.1 Two-Photon Excited Fluorescence (TPEF) Cross-section 16.2 Pulsed Excitation 16.3 Two-Photon Excitation Volume 16.3.1 Gaussian–Lorentzian Volume 16.3.2 3D-Gaussian Volume 16.4 Two-Photon Scanning Microscopy 16.5 Broadband Excitation 16.6 Temporal Focusing 16.7 Multiphoton Microscopy 16.7.1 Advantages of Multiphoton Microscopy 16.8 Problems References 17 Multiharmonic Microscopy 17.1 Nonlinear Susceptibilities 17.1.1 Coherent Interactions 17.1.2 Radiation Patterns 17.2 Second-Harmonic Generation (SHG) Microscopy 17.2.1 Symmetry Properties 17.2.2 3D-Gaussian Approximation 17.2.3 Applications 17.3 Third-Harmonic Generation (THG) Microscopy 17.4 Problems References 18 Pump-Probe Microscopy 18.1 Raman Microscopy 18.1.1 Incoherent Raman Microscopy 18.1.2 Coherent Raman Microscopy 18.2 Brillouin Microscopy 18.3 Photothermal Microscopy 18.4 Photoacoustic Imaging 18.5 Problems References 19 Superresolution 19.1 Restricted Superresolution 19.1.1 PSF Engineering 19.1.2 Synthetic Aperture Holography 19.1.3 Structured Illumination Microscopy 19.2 Unrestricted Superresolution 19.2.1 Nonlinear Structured Illumination Microscopy 19.2.2 Stimulated Emission Depletion (STED) Microscopy 19.2.3 Molecular Switching 19.3 Resolution Versus Localization 19.4 Problems References 20 Imaging in Scattering Media 20.1 Radiative Transfer Equation 20.2 Diffusive Limit 20.3 Small Scattering Angles 20.3.1 Scattering Filter Function 20.4 Propagation of Intensity and Coherence 20.4.1 Plane-Wave Beam 20.4.2 Focused Beam 20.5 Connection with Index of Refraction 20.6 Adaptive Optics 20.6.1 Focus Enhancement 20.6.2 Deep Focusing 20.7 Problems References Appendix A Properties of Fourier Transforms A.1 Fourier Transform Properties A.2 Fourier Transform Theorems A.3 Cylindrical Coordinates A.4 Spherical Symmetry Appendix B Miscellaneous Math B.1 Green Theorem B.2 A Useful Integral B.3 Symmetry Properties of Hermitian and Anti-Hermitian Functions B.4 Properties of Variances B.5 Property of Gaussian Random Variables Appendix C Jones Matrix Description of Polarizers Index