Details

Fundamentals of Light Microscopy and Electronic Imaging


Fundamentals of Light Microscopy and Electronic Imaging


2. Aufl.

von: Douglas B. Murphy, Michael W. Davidson

147,99 €

Verlag: Wiley-Blackwell
Format: PDF
Veröffentl.: 20.09.2012
ISBN/EAN: 9781118382912
Sprache: englisch
Anzahl Seiten: 560

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Beschreibungen

<p><i>Fundamentals of Light Microscopy and Electronic Imaging, Second Edition</i> 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.<br /><br />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.</p> <p>PowerPoint slides of the figures as well as other supplementary materials for instructors are available at a companion website:</p> <p><a href="http://www.wiley.com/go/murphy/lightmicroscopy">www.wiley.com/go/murphy/lightmicroscopy</a></p>
<p>Preface xi</p> <p>Acknowledgments xii</p> <p><b>1. Fundamentals of Light Microscopy 1</b></p> <p>Overview 1</p> <p>Optical Components of the Light Microscope 1</p> <p>Aperture and Image Planes in a Focused, Adjusted Microscope 5</p> <p>Note: Objectives, Eyepieces, and Eyepiece Telescopes 6</p> <p>Koehler Illumination 9</p> <p>Adjusting the Microscope for Koehler Illumination 9</p> <p>Note: Summary of Steps for Koehler Illumination 11</p> <p>Note: Focusing Oil Immersion Objectives 14</p> <p>Fixed Tube Length versus Infinity Optical Systems 15</p> <p>Precautions for Handling Optical Equipment 16</p> <p>Care and Maintenance of the Microscope 17</p> <p>Exercise: Calibration of Magnification 17</p> <p><b>2. Light and Color 21</b></p> <p>Overview 21</p> <p>Light as a Probe of Matter 21</p> <p>The Dual Particle- and Wave-Like Nature of Light 25</p> <p>The Quality of Light 26</p> <p>Properties of Light Perceived by the Eye 27</p> <p>Physical Basis for Visual Perception and Color 28</p> <p>Addition and Subtraction Colors 30</p> <p>Exercise: Complementary Colors 32</p> <p><b>3. Illuminators, Filters, and the Isolation of Specific Wavelengths 35</b></p> <p>Overview 35</p> <p>Illuminators and Their Spectra 35</p> <p>Illuminator Alignment and Bulb Replacement 41</p> <p>Demonstration: Spectra of Common Light Sources 41</p> <p>Demonstration: Aligning a 100-W Mercury Arc Lamp in an Epi-Illuminator 43</p> <p>Filters for Adjusting the Intensity and Wavelength of Illumination 45</p> <p>Effects of Light on Living Cells 50</p> <p><b>4. Lenses and Geometrical Optics 53</b></p> <p>Overview 53</p> <p>Reflection and Refraction of Light 53</p> <p>Image Formation by a Simple Lens 56</p> <p>Note: Real and Virtual Images 57</p> <p>Rules of Ray Tracing for a Simple Lens 58</p> <p>Object–Image Math 58</p> <p>The Principal Aberrations of Lenses 62</p> <p>Designs and Specifications of Objectives 65</p> <p>Condensers 71</p> <p>Oculars 72</p> <p>Microscope Slides and Coverslips 73</p> <p>The Care and Cleaning of Optics 73</p> <p>Exercise: Constructing and Testing an Optical Bench Microscope 76</p> <p><b>5. Diffraction and Interference in Image Formation 79</b></p> <p>Overview 79</p> <p>Diffraction and Interference 80</p> <p>The Diffraction Image of a Point Source of Light 83</p> <p>The Constancy of Optical Path Length between Object and Image 85</p> <p>Demonstration: Viewing the Airy Disk with a Pinhole Aperture 85</p> <p>Effect of Aperture Angle on Diffraction Spot Size 87</p> <p>Diffraction by a Grating and Calculation of Its Line Spacing, <i>D</i> 89</p> <p>Demonstration: The Diffraction Grating 93</p> <p>Abbé’s Theory for Image Formation in the Microscope 94</p> <p>A Diffraction Pattern Is Formed in the Rear Aperture of the Objective 97</p> <p>Demonstration: Observing the Diffraction Image in the Rear Focal Plane of a Lens 98</p> <p>Preservation of Coherence: Essential Requirement for Image Formation 99</p> <p>Exercise: Diffraction by Microscope Specimens 101</p> <p><b>6. Diffraction and Spatial Resolution 103</b></p> <p>Overview 103</p> <p>Numerical Aperture 103</p> <p>Spatial Resolution 105</p> <p>Depth of Field and Depth of Focus 109</p> <p>Optimizing the Microscope Image: A Compromise between Spatial Resolution and Contrast 109</p> <p>Exercise: Resolution of Striae in Diatoms 112</p> <p><b>7. Phase Contrast Microscopy and Darkfield Microscopy 115</b></p> <p>Overview 115</p> <p>Phase Contrast Microscopy 115</p> <p>The Behavior of Waves from Phase Objects in Brightfield Microscopy 119</p> <p>Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry 128</p> <p>Darkfield Microscopy 129</p> <p>Exercise: Darkfield Microscopy 133</p> <p><b>8. Properties of Polarized Light 135</b></p> <p>Overview 135</p> <p>The Generation of Polarized Light 135</p> <p>Demonstration: Producing Polarized Light with a Polaroid Filter 137</p> <p>Polarization by Reflection and Scattering 139</p> <p>Vectorial Analysis of Polarized Light Using a Dichroic Filter 139</p> <p>Double Refraction in Crystals 142</p> <p>Demonstration: Double Refraction by a Calcite Crystal 144</p> <p>Kinds of Birefringence 145</p> <p>Propagation of O and E Wavefronts in a Birefringent Crystal 146</p> <p>Birefringence in Biological Specimens 148</p> <p>Generation of Elliptically Polarized Light by Birefringent Specimens 149</p> <p><b>9. Polarization Microscopy 153</b></p> <p>Overview 153</p> <p>Optics of the Polarizing Microscope 155</p> <p>Adjusting the Polarizing Microscope 156</p> <p>Appearance of Birefringent Objects in Polarized Light 157</p> <p>Principles of Action of Retardation Plates and Three Popular Compensators 158</p> <p>Demonstration: Making a <i>λ</i>-Plate from a Piece of Cellophane 162</p> <p>Exercise: Determination of Molecular Organization in Biological Structures Using a Full Wave Plate Compensator 167</p> <p><b>10. Differential Interference Contrast Microscopy and Modulation Contrast Microscopy 173</b></p> <p>Overview 173</p> <p>The DIC Optical System 173</p> <p>Demonstration: The Action of a Wollaston Prism in Polarized Light 179</p> <p>Modulation Contrast Microscopy 190</p> <p>Exercise: DIC Microscopy 194</p> <p><b>11. Fluorescence Microscopy 199</b></p> <p>Overview 199</p> <p>Applications of Fluorescence Microscopy 201</p> <p>Physical Basis of Fluorescence 202</p> <p>Properties of Fluorescent Dyes 205</p> <p>Demonstration: Fluorescence of Chlorophyll and Fluorescein 206</p> <p>Autofluorescence of Endogenous Molecules 211</p> <p>Demonstration: Fluorescence of Biological Materials under UV Light 213</p> <p>Fluorescent Dyes and Proteins in Fluorescence Microscopy 213</p> <p>Arrangement of Filters and the Epi-Illuminator in the Fluorescence Microscope 218</p> <p>Objectives and Spatial Resolution in Fluorescence Microscopy 224</p> <p>Causes of High Fluorescence Background 225</p> <p>The Problem of Bleedthrough with Multiply Stained Specimens 227</p> <p>Quenching, Blinking, and Photobleaching 228</p> <p>Examining Fluorescent Molecules in Living Cells 230</p> <p><b>12. Fluorescence Imaging of Dynamic Molecular Processes 233</b></p> <p>Overview 233</p> <p>Modes of Dynamic Fluorescence Imaging 234</p> <p>Förster Resonance Energy Transfer 236</p> <p>Applications 244</p> <p>Fluorescence Recovery after Photobleaching 245</p> <p>TIRF Microscopy: Excitation by an Evanescent Wave 252</p> <p>Advanced and Emerging Dynamic Fluoresence Techniques 261</p> <p><b>13. Confocal Laser Scanning Microscopy 265</b></p> <p>Overview 265</p> <p>The Optical Principle of Confocal Imaging 267</p> <p>Demonstration: Isolation of Focal Plane Signals with a Confocal Pinhole 271</p> <p>Advantages of CLSM over Widefield Fluorescence Systems 273</p> <p>Criteria Defining Image Quality and the Performance of an Electronic Imaging System 275</p> <p>Confocal Adjustments and Their Effects on Imaging 277</p> <p>Photobleaching 286</p> <p>General Procedure for Acquiring a Confocal Image 286</p> <p>Performance Check of a Confocal System 288</p> <p>Fast (Real-Time) Imaging in Confocal Microscopy 288</p> <p>Spectral Analysis: A Valuable Enhancement for Confocal Imaging 295</p> <p>Optical Sectioning by Structured Illumination 297</p> <p>Deconvolution Microscopy 298</p> <p>Exercise: Effect of Confocal Variables on Image Quality 304</p> <p><b>14. Two-photon Excitation Fluorescence Microscopy 307</b></p> <p>Overview 307</p> <p>The Problem of Photon Scattering in Deep Tissue Imaging 308</p> <p>Two-Photon Excitation Is a Nonlinear Process 309</p> <p>Localization of Excitation 314</p> <p>Why Two-Photon Imaging Works 317</p> <p>Resolution 318</p> <p>Equipment 319</p> <p>Three-Photon Excitation 325</p> <p>Second Harmonic Generation Microscopy 326</p> <p><b>15. Superresolution Imaging 331</b></p> <p>Overview 331</p> <p>The RESOLFT Concept 333</p> <p>Single-Molecule Localization Microscopy 334</p> <p>Structured Illumination Microscopy 343</p> <p>Stimulated Emission Depletion (STED) Microscopy: Superresolution by PSF Engineering 349</p> <p><b>16. Imaging Living Cells with the Microscope 357</b></p> <p>Overview 357</p> <p>Labeling Strategies for Live-Cell Imaging 358</p> <p>Control of Illumination 361</p> <p>Control of Environmental Conditions 365</p> <p>Optics, Detectors, and Hardware 372</p> <p>Evaluating Live-Cell Imaging Results 384</p> <p>Exercise: Fluorescence Microscopy of Living Tissue Culture Cells 384</p> <p><b>17. Fundamentals of Digital Imaging 389</b></p> <p>Overview 389</p> <p>The Charge-Coupled Device (CCD Imager) 390</p> <p>CCD Designs 396</p> <p>Note: Interline CCD Imagers: The Design of Choice for Biomedical Imaging 398</p> <p>Back-Thinned Sensors 398</p> <p>EMCCD Cameras: High Performance Design for Greatest Sensitivity 399</p> <p>Scientific CMOS: The Next Generation of Scientific Imagers 400</p> <p>Camera Variables Affecting CCD Readout and Image Quality 401</p> <p>Six Terms Define Imaging Performance 404</p> <p>Aliasing 409</p> <p>Color Cameras 410</p> <p>Exercise: Evaluating the Performance of a CCD Camera 411</p> <p><b>18. Digital Image Processing 415</b></p> <p>Overview 415</p> <p>Preliminaries: Image Display and Data Types 416</p> <p>Histogram Adjustment 417</p> <p>Adjusting Gamma (<i>γ</i>) to Create Exponential LUTs 421</p> <p>Flat-Field Correction 421</p> <p>Image Processing With Filters 425</p> <p>Signal-to-Noise Ratio 432</p> <p>The Use of Color 438</p> <p>Images as Research Data and Requirements for Scientific Publication 442</p> <p>Exercise: Flat-Field Correction and Determination of S/N Ratio 448</p> <p>Appendix A: Answer Key to Exercises 451</p> <p>Appendix B: Materials for Demonstrations and Exercises 455</p> <p>Appendix C: Sources of Materials for Demonstrations and Exercises 463</p> <p>Glossary 465</p> <p>Microscopy Web Resources 509</p> <p>Recommended Reading 521</p> <p>References 523</p> <p>Index 531</p>
<p>“This should be provided to all beginning graduate students entering microscopy labs. It describes the complicated hardware of the system, while also explaining the physics principles of microscopy on a simplistic level for basic biologists. The authors achieve a perfect balance of theory and methods.”  (<i>Doody’s</i>, 15 November 2013)</p> <p>“It should be particularly useful to researchers getting started in the field of microscopy as well as seasoned professionals. Summing Up: Highly recommended. Graduate students, researchers/faculty, and professionals/practitioners.”  (<i>Choice</i>, 1 October 2013)</p> <p>“In summary, Fundamentals of Light Microscopy, Second Edition is a recommended starting point for the novice in microscopy and electronic imaging.”  (<i>Journal of Biomedical Optics</i>, 1 February 2013)</p>
<p><b>DOUGLAS B. MURPHY</b> 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.</p> <p><b>MICHAEL W. DAVIDSON</b> 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.</p>
<p>"This book will provide individuals without background knowledge in optical physics, electronics, or image processing with many of the basic facts they need to know to understand both the power and limitations of their images."<br /> <i>—Cell Biology Education</i> on the First Edition</p> <p><i>Fundamentals of Light Microscopy and Electronic Imaging, Second Edition</i> 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.</p> <p>Written in simple, clear language, 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. Including new sections on live cell imaging, measurement of protein dynamics, deconvolution, multiphoton microscopy, and superresolution microscopy, <i>Fundamentals of Light Microscopy and Electronic Imaging, Second Edition</i> features the following chapters:</p> <ul> <li>Fundamentals of Light Microscopy</li> <li>Light and Color</li> <li>Illuminators, Filters, and the Isolation of Specific Wavelengths</li> <li>Lenses and Geometrical Optics</li> <li>Diffraction and Interference in Image Formation</li> <li>Diffraction and Spatial Resolution</li> <li>Phase Contrast Microscopy and Darkfield Microscopy</li> <li>Properties of Polarized Light</li> <li>Polarization Microscopy</li> <li>Differential Interference Contrast (DIC) Microscopy and Modulation Contrast Microscopy</li> <li>Fluorescence Microscopy</li> <li>Fluorescence Imaging of Dynamic Molecular Processes</li> <li>Confocal Laser Scanning Microscopy</li> <li>Two-Photon Excitation Fluorescence Microscopy</li> <li>Superresolution Imaging</li> <li>Imaging Living Cells with the Microscope</li> <li>Fundamentals of Digital Imaging</li> <li>Digital Imaging Processing</li> </ul>

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