Douglas B. Murphy, Michael W. Davidson

Fundamentals of Light Microscopy and Electronic Imaging (eBook, PDF)

• Format: PDF

Produktbeschreibung

Fundamentals of Light Microscopy and Electronic Imaging, Second Edition provides a coherent introduction to the principles and applications of the integrated optical microscope system, covering both theoretical and practical considerations. It expands and updates discussions of multi-spectral imaging, intensified digital cameras, signal colocalization, and uses of objectives, and offers guidance in the selection of microscopes and electronic cameras, as well as appropriate auxiliary optical systems and fluorescent tags. The book is divided into three sections covering optical principles in diffraction and image formation, basic modes of light microscopy, and components of modern electronic imaging systems and image processing operations. Each chapter introduces relevant theory, followed by descriptions of instrument alignment and image interpretation. This revision includes new chapters on live cell imaging, measurement of protein dynamics, deconvolution microscopy, and interference microscopy. PowerPoint slides of the figures as well as other supplementary materials for instructors are available at a companion website: www.wiley.com/go/murphy/lightmicroscopy

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Produktdetails

• Verlag: John Wiley & Sons
• Seitenzahl: 560
• Erscheinungstermin: 20. September 2012
• Englisch
• ISBN-13: 9781118382912
• Artikelnr.: 37345310

Autorenporträt

DOUGLAS B. MURPHY supervises core facilities in microscopy and histology at the new HHMI Janelia Farm Research Campus in Ashburn, Virginia. An Adjunct Professor of Cell Biology at Johns Hopkins School of Medicine in Baltimore, Maryland, Dr. Murphy helped establish the School of Medicine Microscope Facility there, which he supervised until 2006. MICHAEL W. DAVIDSON is an assistant scholar/scientist affiliated with the National High Magnetic Field Laboratory and the Department of Biological Science at Florida State University where he is involved in developing educational websites. His digital images and photomicrographs have graced the covers of over 2,000 publications.

Inhaltsangabe

Preface xi Acknowledgments xii 1. FUNDAMENTALS OF LIGHT MICROSCOPY 1
Overview 1 Optical Components of the Light Microscope 1 Aperture and Image
Planes in a Focused, Adjusted Microscope 5 Note: Objectives, Eyepieces, and
Eyepiece Telescopes 6 Koehler Illumination 9 Adjusting the Microscope for
Koehler Illumination 9 Note: Summary of Steps for Koehler Illumination 11
Note: Focusing Oil Immersion Objectives 14 Fixed Tube Length versus Infi
nity Optical Systems 15 Precautions for Handling Optical Equipment 16 Care
and Maintenance of the Microscope 17 Exercise: Calibration of Magnification
17 2. LIGHT AND COLOR 21 Overview 21 Light as a Probe of Matter 21 The Dual
Particle- and Wave-Like Nature of Light 25 The Quality of Light 26
Properties of Light Perceived by the Eye 27 Physical Basis for Visual
Perception and Color 28 Addition and Subtraction Colors 30 Exercise:
Complementary Colors 32 3. ILLUMINATORS, FILTERS, AND THE ISOLATION OF
SPECIFIC WAVELENGTHS 35 Overview 35 Illuminators and Their Spectra 35
Illuminator Alignment and Bulb Replacement 41 Demonstration: Spectra of
Common Light Sources 41 Demonstration: Aligning a 100-W Mercury Arc Lamp in
an Epi-Illuminator 43 Filters for Adjusting the Intensity and Wavelength of
Illumination 45 Effects of Light on Living Cells 50 4. LENSES AND
GEOMETRICAL OPTICS 53 Overview 53 Reflection and Refraction of Light 53
Image Formation by a Simple Lens 56 Note: Real and Virtual Images 57 Rules
of Ray Tracing for a Simple Lens 58 Object-Image Math 58 The Principal
Aberrations of Lenses 62 Designs and Specifi cations of Objectives 65
Condensers 71 Oculars 72 Microscope Slides and Coverslips 73 The Care and
Cleaning of Optics 73 Exercise: Constructing and Testing an Optical Bench
Microscope 76 5. DIFFRACTION AND INTERFERENCE IN IMAGE FORMATION 79
Overview 79 Diffraction and Interference 80 The Diffraction Image of a
Point Source of Light 83 The Constancy of Optical Path Length between
Object and Image 85 Demonstration: Viewing the Airy Disk with a Pinhole
Aperture 85 Effect of Aperture Angle on Diffraction Spot Size 87
Diffraction by a Grating and Calculation of Its Line Spacing, D 89
Demonstration: The Diffraction Grating 93 Abbé's Theory for Image Formation
in the Microscope 94 A Diffraction Pattern Is Formed in the Rear Aperture
of the Objective 97 Demonstration: Observing the Diffraction Image in the
Rear Focal Plane of a Lens 98 Preservation of Coherence: Essential
Requirement for Image Formation 99 Exercise: Diffraction by Microscope
Specimens 101 6. DIFFRACTION AND SPATIAL RESOLUTION 103 Overview 103
Numerical Aperture 103 Spatial Resolution 105 Depth of Field and Depth of
Focus 109 Optimizing the Microscope Image: A Compromise between Spatial
Resolution and Contrast 109 Exercise: Resolution of Striae in Diatoms 112
7. PHASE CONTRAST MICROSCOPY AND DARKFIELD MICROSCOPY 115 Overview 115
Phase Contrast Microscopy 115 The Behavior of Waves from Phase Objects in
Brightfi eld Microscopy 119 Exercise: Determination of the Intracellular
Concentration of Hemoglobin in Erythrocytes by Phase Immersion
Refractometry 128 Darkfi eld Microscopy 129 Exercise: Darkfi eld Microscopy
133 8. PROPERTIES OF POLARIZED LIGHT 135 Overview 135 The Generation of
Polarized Light 135 Demonstration: Producing Polarized Light with a
Polaroid Filter 137 Polarization by Refl ection and Scattering 139
Vectorial Analysis of Polarized Light Using a Dichroic Filter 139 Double
Refraction in Crystals 142 Demonstration: Double Refraction by a Calcite
Crystal 144 Kinds of Birefringence 145 Propagation of O and E Wavefronts in
a Birefringent Crystal 146 Birefringence in Biological Specimens 148
Generation of Elliptically Polarized Light by Birefringent Specimens 149 9.
POLARIZATION MICROSCOPY 153 Overview 153 Optics of the Polarizing
Microscope 155 Adjusting the Polarizing Microscope 156 Appearance of
Birefringent Objects in Polarized Light 157 Principles of Action of
Retardation Plates and Three Popular Compensators 158 Demonstration: Making
a lambda-Plate from a Piece of Cellophane 162 Exercise: Determination of
Molecular Organization in Biological Structures Using a Full Wave Plate
Compensator 167 10. DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY AND
MODULATION CONTRAST MICROSCOPY 173 Overview 173 The DIC Optical System 173
Demonstration: The Action of a Wollaston Prism in Polarized Light 179
Modulation Contrast Microscopy 190 Exercise: DIC Microscopy 194 11.
FLUORESCENCE MICROSCOPY 199 Overview 199 Applications of Fluorescence
Microscopy 201 Physical Basis of Fluorescence 202 Properties of Fluorescent
Dyes 205 Demonstration: Fluorescence of Chlorophyll and Fluorescein 206
Autofl uorescence of Endogenous Molecules 211 Demonstration: Fluorescence
of Biological Materials under UV Light 213 Fluorescent Dyes and Proteins in
Fluorescence Microscopy 213 Arrangement of Filters and the Epi-Illuminator
in the Fluorescence Microscope 218 Objectives and Spatial Resolution in
Fluorescence Microscopy 224 Causes of High Fluorescence Background 225 The
Problem of Bleedthrough with Multiply Stained Specimens 227 Quenching,
Blinking, and Photobleaching 228 Examining Fluorescent Molecules in Living
Cells 230 12. FLUORESCENCE IMAGING OF DYNAMIC MOLECULAR PROCESSES 233
Overview 233 Modes of Dynamic Fluorescence Imaging 234 Förster Resonance
Energy Transfer 236 Applications 244 Fluorescence Recovery after
Photobleaching 245 TIRF Microscopy: Excitation by an Evanescent Wave 252
Advanced and Emerging Dynamic Fluoresence Techniques 261 13. CONFOCAL LASER
SCANNING MICROSCOPY 265 Overview 265 The Optical Principle of Confocal
Imaging 267 Demonstration: Isolation of Focal Plane Signals with a Confocal
Pinhole 271 Advantages of CLSM over Widefield Fluorescence Systems 273
Criteria Defining Image Quality and the Performance of an Electronic
Imaging System 275 Confocal Adjustments and Their Effects on Imaging 277
Photobleaching 286 General Procedure for Acquiring a Confocal Image 286
Performance Check of a Confocal System 288 Fast (Real-Time) Imaging in
Confocal Microscopy 288 Spectral Analysis: A Valuable Enhancement for
Confocal Imaging 295 Optical Sectioning by Structured Illumination 297
Deconvolution Microscopy 298 Exercise: Effect of Confocal Variables on
Image Quality 304 14. TWO-PHOTON EXCITATION FLUORESCENCE MICROSCOPY 307
Overview 307 The Problem of Photon Scattering in Deep Tissue Imaging 308
Two-Photon Excitation Is a Nonlinear Process 309 Localization of Excitation
314 Why Two-Photon Imaging Works 317 Resolution 318 Equipment 319
Three-Photon Excitation 325 Second Harmonic Generation Microscopy 326 15.
SUPERRESOLUTION IMAGING 331 Overview 331 The RESOLFT Concept 333
Single-Molecule Localization Microscopy 334 Structured Illumination
Microscopy 343 Stimulated Emission Depletion (STED) Microscopy:
Superresolution by PSF Engineering 349 16. IMAGING LIVING CELLS WITH THE
MICROSCOPE 357 Overview 357 Labeling Strategies for Live-Cell Imaging 358
Control of Illumination 361 Control of Environmental Conditions 365 Optics,
Detectors, and Hardware 372 Evaluating Live-Cell Imaging Results 384
Exercise: Fluorescence Microscopy of Living Tissue Culture Cells 384 17.
FUNDAMENTALS OF DIGITAL IMAGING 389 Overview 389 The Charge-Coupled Device
(CCD Imager) 390 CCD Designs 396 Note: Interline CCD Imagers: The Design of
Choice for Biomedical Imaging 398 Back-Thinned Sensors 398 EMCCD Cameras:
High Performance Design for Greatest Sensitivity 399 Scientific CMOS: The
Next Generation of Scientific Imagers 400 Camera Variables Affecting CCD
Readout and Image Quality 401 Six Terms Define Imaging Performance 404
Aliasing 409 Color Cameras 410 Exercise: Evaluating the Performance of a
CCD Camera 411 18. DIGITAL IMAGE PROCESSING 415 Overview 415 Preliminaries:
Image Display and Data Types 416 Histogram Adjustment 417 Adjusting Gamma
(gamma) to Create Exponential LUTs 421 Flat-Field Correction 421 Image
Processing With Filters 425 Signal-to-Noise Ratio 432 The Use of Color 438
Images as Research Data and Requirements for Scientific Publication 442
Exercise: Flat-Field Correction and Determination of S/N Ratio 448 Appendix
A: Answer Key to Exercises 451 Appendix B: Materials for Demonstrations and
Exercises 455 Appendix C: Sources of Materials for Demonstrations and
Exercises 463 Glossary 465 Microscopy Web Resources 509 Recommended Reading
521 References 523 Index 531

Rezensionen

"In summary, Fundamentals of Light Microscopy, Second Edition is a recommended starting point for the novice in microscopy and electronic imaging." (Journal of Biomedical Optics, 1 February 2013)