Optical imaging techniques in cell biology / [edited by] Guy Cox.

Format:

Book

Contributor:

Cox, Guy, 1945-

EBSCOhost.

Rudolph G. Schmieder Fund.

Language:

English

Subjects (All):

Cytology.

Imaging systems in biology.

Cytological Techniques--methods.

Microscopy--methods.

Medical Subjects:

Cytological Techniques--methods.

Microscopy--methods.

Physical Description:

1 online resource (xvii, 297 pages) : illustrations (some color)

Edition:

Second edition.

Place of Publication:

Boca Raton, FL : CRC Press, [2012]

System Details:

text file

Summary:

Microscopy specialists Cox, Teresa Dibbayawan, Eleanor Kable (allU. of Sydney), and Nuno Moreno (Instituto Gulbenkain de Ciência,Portugal) describe how visible light is still used in cell biologyalongside all the fancy new imaging technologies. Among their topicsare the light microscope, image capture, the confocal microscope,aberrations and their consequences, deconvolution and imageprocessing, stereoscopy and reconstruction for three-dimensionalimaging, fluorescent staining, evanescent wave microscopy, and beyondthe diffraction limit. Appendices provide practical advice such asmicroscope care and maintenance, and keeping cells alive under themicroscope. The first edition was published in 2007. Annotation ©2012 Book News, Inc., Portland, OR (booknews.com)

Contents:

Chapter 1 The Light Microscope 1

Lenses and Microscopes 1

The Back Focal Plane of a Lens 4

Good Resolution 8

Resolution: Rayleigh's Approach 9

Abbe 13

Add a Drop of Oil 16

Köhler Illumination 18

References 19

Chapter 2 Optical Contrasting Techniques 21

Darkfield 21

Phase Contrast 23

Polarization 25

Differential Interference Contrast 28

Hoffman Modulation Contrast 31

Which Technique Is Best? 34

References 34

Chapter 3 Fluorescence and Fluorescence Microscopy 35

What Is Fluorescence? 35

What Makes a Molecule Fluorescent? 39

The Fluorescence Microscope 40

Optical Arrangement 40

Light Source 40

Filter Sets: Excitation Filter, Dichroic Mirror, and Barrier Filter 43

References 47

Chapter 4 Image Capture 49

Optical Layout for Image Capture 49

Color Recording 49

Additive Color Model 50

Subtractive Color Model 51

CCD Cameras 53

Frame-Transfer Array 55

Interline-Transfer Array 55

Back Illumination 56

Binning 56

Capturing Color 57

Filter Wheels 57

Filter Mosaics 58

Three CCD Elements with Dichroic Beamsplitters 58

Boosting the Signal 58

References 61

Chapter 5 The Confocal Microscope 63

The Scanning Optical Microscope 63

The Confocal Principle 63

Resolution and Point Spread Function 65

Lateral Resolution in the Confocal Microscope 68

Practical Confocal Microscopes 69

The Light Source: Lasers 71

Gas Lasers 72

Solid-State Lasers 72

Semiconductor Lasers 73

Supercontinuum Lasers 74

Laser Delivery 75

The Primary Beamsplitter 76

Beam Scanning 77

Pinhole and Signal Channel Configurations 77

Detectors 81

References 82

Further Reading 82

Chapter 6 The Digital Image 83

Pixels and Voxels 83

Contrast 83

Spatial Sampling: The Nyquist Criterion 85

Temporal Sampling: Signal-to-Noise Ratio 86

Multichannel Images 89

References 91

Further Reading 91

Chapter 7 Aberrations and Their Consequences 93

Geometrical Aberrations 93

Spherical Aberration 93

Coma 99

Astigmatism 100

Field Curvature 100

Chromatic Aberration 102

Chromatic Difference of Magnification 103

Practical Consequences 105

Apparent Depth 107

References 108

Further Reading 108

Chapter 8 Nonlinear Microscopy 109

Multiphoton Microscopy 109

Principles of Two-Photon Fluorescence 109

Theory and Practice 111

Lasers for Nonlinear Microscopy 112

Advantages of Two-Photon Excitation 114

Construction of a Multiphoton Microscope 115

Fluorochromes for Multiphoton Microscopy 116

Second Harmonic Microscopy 117

Summary 122

References 122

Further Reading 123

Chapter 9 High-Speed Confocal Microscopy 125

Tandem Scanning (Spinning Disk) Microscopes 125

Petràn System 125

One-Sided Tandem Scanning Microscopes (OTSMS) 128

Microlens Array: The Yokogawa System 128

Slit-Scanning Microscopes 130

Multipoint-Array Scanners 132

Structured Illumination 135

References 135

Further Reading 136

Chapter 10 Deconvolution and Image Processing 137

Deconvolution 137

Deconvolving Confocal Images 142

Image Processing 142

Grayscale Operations 143

Image Arithmetic 145

Convolution: Smoothing and Sharpening 146

References 149

Further-Reading 149

Chapter 11 Three-Dimensional Imaging: Stereoscopy and Reconstruction 151

Surfaces: Two-and-a-Half Dimensions 151

Perception of the 3D World 153

Motion Parallax 153

Convergence and Focus of Our Eyes 153

Perspective 154

Concealment of One Object by Another 154

Our Knowledge of the Size and Shape of Everyday Things 154

Light and Shade 154

Limitations of Confocal Microscopy 154

Stereoscopy 155

Three-Dimensional Reconstruction 157

Techniques That Require Identification of "Objects" 158

Techniques That Create Views Directly from Intensity Data 161

Simple Projections 161

Weighted Projection (Alpha Blending) 163

References 168

Chapter 12 Green Fluorescent Protein 169

Structure and Properties of GFP 169

GFP Variants 170

Applications of GFP 175

Heat Shock 176

Cationic Lipid Reagents 176

DEAE-Dextran and Polybrene 176

Calcium Phosphate Coprecipitation 176

Electroporation 177

Microinjection 177

Gene Gun 177

Plants: Agrobacterium 179

References 1801

Chapter 13 Fluorescent Staining / Teresa Dibbayawan Dibbayawan, Teresa, Eleanor Kable Kable, Eleanor, Guy Cox Cox, Guy 181

Immunolabeling 181

Types of Antibody 182

Raising Antibodies 183

Labeling 185

Fluorescent Stains for Cell Components and Compartments 187

References 190

Chapter 14 Quantitative Fluorescence 191

Fluorescence Intensity Measurements 191

Linearity Calibration 191

Measurement 192

Colocalization 192

Ratio Imaging 195

Cell Loading 199

Membrane Potential 199

Fast-Response Dyes 200

Slow-Response Dyes 200

Fluorescence Recovery after Photobleaching 201

References 205

Chapter 15 Advanced Fluorescence Techniques: FLIM, FRET, and FCS 207

Fluorescence Lifetime 207

Practical Lifetime Microscopy (FLIM) 209

Frequency Domain 209

Time Domain 209

Fluorescence Resonant Energy Transfer (FRET) 213

Why Use FRET? 214

Identifying and Quantifying FRET 215

Increase in Brightness of Acceptor Emission 215

Quenching of Emission from the Donor 219

Lifetime of Donor Emission 220

Protection from Bleaching of Donor 221

Fluorescence Correlation Spectroscopy (FCS) 223

Raster Image Correlation Spectroscopy 224

References 225

Further Reading 226

Chapter 16 Evanescent Wave Microscopy 227

The Near-Field and Evanescent Waves 227

Total Internal Reflection Microscopy 227

Near-Field Microscopy 230

References 236

Chapter 17 Beyond the Diffraction Limit 237

4Pi and Multiple-Objective Microscopy 237

Stimulated Emission Depletion (STED) 240

Structured Illumination 245

Stochastic Techniques 247

Super-Resolution Summary 252

References 253.

Notes:

Description based on print version record.

Rev. ed. of: Optical imaging techniques in cell biology / Guy Cox. c2007.

Includes bibliographical references and index.

Electronic reproduction. Ipswich, MA Available via World Wide Web.

Local Notes:

Acquired for the Penn Libraries with assistance from the Rudolph G. Schmieder Fund.

Related Work:

Cox, Guy, 1945- Optical imaging techniques in cell biology.

ISBN:

1439848289

9781439848289

Publisher Number:

99954676811

Access Restriction:

Restricted for use by site license.