Fundamentals of Light Microscopy and Electronic Imaging

2. Revised Auflage 2012
ISBN: 978-0-471-69214-0
Verlag: Wiley

Buch, Englisch, 560 Seiten, Format (B × H): 181 mm x 264 mm, Gewicht: 1451 g

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

Autoren/Hrsg.

Murphy, Douglas B

Davidson, Michael W

Fachgebiete

• Naturwissenschaften Biowissenschaften Molekularbiologie
• Naturwissenschaften Biowissenschaften Zellbiologie
• Naturwissenschaften Physik Elektromagnetismus Mikroskopie, Spektroskopie

Inhaltsverzeichnis

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 Infinity 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 Specifications 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 Brightfield Microscopy 119

Exercise: Determination of the Intracellular Concentration of Hemoglobin in Erythrocytes by Phase Immersion Refractometry 128

Darkfield Microscopy 129

Exercise: Darkfield 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 Reflection and Scattering 139

Vectorial Analysis of Polarized Light Using a Dichroic Filter 139

Double Refraction in Crystals 142

Über Autor(innen)

Davidson, Michael W
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.

Murphy, Douglas B
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.

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.