Embed Size (px)
Citation preview
Light and electron microscopy
Elizabeth M. Slayter formerly of Brandeis University
Henry S. Slayter Harvard Medical School and Dana-Farber Cancer Institute
CAMBRIDGE UNIVERSITY PRESS
Contents
Preface page xiii List of abbreviations xv
1 Introduction 1 1.1 Magnification, resolution, and contrast 1 1.2 Microscopes: definitions and brief history 2 1.3 Microscope design 4 1.4 Mathematical aspects 6
2 Light and electrons 7 2.1 The nature of light 7 2.2 Geometrical optics 10
2.2.1 Reflection and refraction of light 10 2.3 Wave optics ("physical optics") 12
2.3.1 Simple harmonic motion 13 2.3.2 Wave equations 15
2.4 Characteristics of wave motions 15 2.4.1 Amplitude and intensity 15 2.4.2 Wavelength, frequency, and the electromagnetic spectrum 16
2.4.2.1 Electromagnetic radiation 16 2.4.2.2 Electrons 17 2.4.2.3 The electromagnetic spectrum 17
2.4.3 Phase 19 2.4.4 Monochromaticity 19 2.4.5 Coherence 20 2.4.6 Plane of polarization 20
2.5 Limits of Ijght-electron parallelism 21
3 Wave interactions 23 3.1 The principle of superposition 23 3.2 Huygens' principle 24 3.3 Resultant vibrations 25
3.3.1 Graphic computation of resultant waveforms 25 3.3.2 Algebraic computation of resultant waveforms 26 3.3.3 Resultant waveforms by vector addition 27 3.3.4 Resultant waveforms by the method of complex
amplitudes 27 3.4 Superposition of waves of different frequencies; Fourier optics 29
3.4.1 Representation of periodic functions as Fourier series 30
v
vi Contents
3.4.2 Representation of nonperiodic functions as Fourier transforms 33
3.4.3 Some properties of Fourier transforms 33 3.5 Applications of Fourier theory to basic properties of radiation 36
3.5.1 Coherence and monochromaticity 36 3.5.2 Phase and group velocities 37
4 Interference effects and diffraction patterns 39 4.1 Opticalpath 39 4.2 Interference and diffraction 40 4.3 Interference effects 40
4.3.1 Interference colors in thin films 40 4.3.2 Lens coatings 42 4.3.3 The interference filter 42
4.4 Diffraction patterns 43 4.4.1 The single-slit diffraction pattern 43 4.4.2 The double-slit diffraction pattern 44 4.4.3 The diffraction grating 45 4.4.4 Diffraction from crystals; the Bragg law 46 4.4.5 The small circular aperture 47
4.5 Holography 49
5 Polarized light 51 5.1 Notation and vectorial representation 51 5.2 Circularly and elliptically polarized light 52 5.3 Interactions of polarized light with oriented matter 56
5.3.1 Anisotropie effects 56 5.3.2 Birefringence 56
5.3.2.1 Theopticaxis 57 5.3.2.2 Ordinary and extraordinary rays 57 5.3.2.3 Sign of birefringence 58 5.3.2.4 Wave surfaces and index ellipsoids 58 5.3.2.5 The passage of O- and E-rays through
birefringent media 59 5.3.2.6 Types of birefringence 61
5.4 Sources of plane-polarized light 61 5.4.1 Dichroic polarizers 61 5.4.2 Birefringent polarizers 61 5.4.3 Polarization by reflection 62
5.5 Polarizers, analyzers, and compensators 63
6 Lenses 65 6.1 The ideal lens 65 6.2 Focusing properties of curved surfaces 66 6.3 Focal points, focal lengths, and focal planes 68 6.4 Thin and thick glass lenses 68 6.5 Magnification 71
Contents vn
6.6 Additional terms used to describe lenses and lens Systems 71 6.7 Image location 73 6.8 Electron lenses 73
6.8.1 Electron trajectories in an axially Symmetrie magnetic field 74
6.8.2 Action of the magnetic lens 76 6.8.3 Construction of magnetic lenses 76
6.9 Lens errors 79 6.9.1 Geometrie or "third-order" errors 79
6.9.1.1 Spherical aberration 80 6.9.1.2 Other geometric errors 82
6.9.2 Chromatic errors 83 6.9.3 Technical limitations 86
Imaging: microscopy and diffraction 88 7.1 Focal planes; formation of a diffraction pattern 88 7.2 The Fourier optical approach to imaging 88
7.2.1 A verbal description of imaging in terms of Fourier optics 89 7.3 Transforms and inverse transforms; the optical diffractometer 91 7.4 Diffraction and reeiprocal space 92 7.5 Imaging and diffraction: the phase problem 93
Contrast 95 8.1 General aspects of image contrast 95 8.2 Light-optical contrast 97
8.2.1 Amplitude contrast 97 8.2.2 Phase contrast 98
8.3 Electron-optical contrast 100 8.3.1 Contrast in transmission microscopy: an overview 101 8.3.2 Electron contrast categories 101 8.3.3 The nature of electron scattering 102
8.3.3.1 Elastic and inelastic scattering 102 8.3.3.2 Rutherford scattering 103 8.3.3.3 Scattering factors and scattering cross sections 105 8.3.3.4 Inelastic scattering and the electron energy-loss
spectrum 107 8.3.4 Electron phase contrast 109
8.3.4.1 Focus fringes and aberration contrast 110 8.3.4.2 Bragg reflections 111
8.3.5 Electron contrast in the scanning electron microscope (SEM) 111
8.4 Contrast transfer funetions 112 8.5 Mass-thickness contrast 116
Resolution 118 9.1 Detection of unresolved objeets 118 9.2 Fourier optics and limiting resolution 119
9.2.1 Images of extremely small objeets 120
viii Contents
9.3 Coherence properties and resolution 121 9.4 Theories of ultimate resolving power 121
9.4.1 Minimum resolvable Separation between incoherently illuminated points 121 9.4.1.1 Approach 121 9.4.1.2 Quantitative evaluation 122
9.4.2 Minimum resolvable spacing in a coherently illuminated specimen 124
9.5 Numerical aperture and the immersion principle 126 9.5.1 Optimal resolution in light-optical Systems 127 9.5.2 The resolving power of electron lenses 127
9.6 Extension of the classical resolution limit 128 9.7 Conflicts between contrast and resolution 129
10 The light microscope 131 10.1 Microscope elements and microscope Systems 131
10.1.1 Light sources 131 10.1.2 The condenser lens 133 10.1.3 The objective lens 134 10.1.4 Mechanical elements 136 10.1.5 The eyepiece lens 137 10.1.6 Stereomicroscopes 139
10.2 Magnification and calibration 139 10.3 Depths of field and focus for light microscopes 140 10.4 Alternative modes of optical microscopy 142
10.4.1 Darkfield 142 10.4.2 Fluorescence microscopy 143 10.4.3 Ultraviolet microscopy 145 10.4.4 Total-internal-reflection microscopy 146
11 Imaging of phase objects 149 11.1 Phase-contrast principles 150 11.2 The phase-contrast microscope 152
11.2.1 Interpretation of the phase-contrast image 153 11.3 The differential-interference-contrast (DIC) microscope 154
11.3.1 Wollaston prisms 154 11.3.2 Elements of the DIC microscope 155 11.3.3 The DIC image 157
11.4 The modulation-contrast microscope (MCM) 158 11.5 Reflection-interference microscopy 160 11.6 Interferometer microscopes 161
11.6.1 The interferometric microscope image 162 11.6.2 Practical Systems 163
11.6.2.1 The Jamin-Lebedeff interferometric microscope 163
11.6.2.2 The film-thickness interferometer using a Fizeau plate 164
Contents ix
11.7 Quantitative intracellular-mass measurements by interferometric microscopy 166
12
13
14
Polar 12.1 12.2 12.3
12.4
12.5
12.6
izing microscopy Applications of the polarizing microscope The polarizing microscope Basic concepts of polarizing microscopy 12.3.1 Concerning terms describing polarization effects 12.3.2 Positions of extinction and maximum brightness 12.3.3 Retardation: slow and fast rays Compensators 12.4.1 Theredlplate 12.4.2 The wedge compensator 12.4.3 The tilting compensator 12.4.4 The quarter-wave plate 12.4.5 The fixed-retardation compensator (Koehler-Brace
method) Biological polarizing microscopes 12.5.1 The polarization cross and diffraction anomalies 12.5.2 Correction of diffraction anomalies Measurements with the polarizing microscope 12.6.1 Location of the optic axis 12.6.2 Determination of the sign and magnitude of
birefringence 12.6.3 Determination of the type of birefringence 12.6.4 Determination of the index ellipsoid
Prospects for biological x-ray microscopy 13.1
13.2 13.3
X-ray imaging 13.1.1 The refraction problem 13.1.2 X-ray contrast X-ray sources Types of x-ray microscopes 13.3.1 Zone-plate instruments 13.3.2 Mirror-lens microscopes 13.3.3 Contact microradiography
The conventional transmission eiectron microscope 14.1 Microscope column elements
14.1.1 Vacuum Systems 14.1.2 The eiectron source 14.1.3 The condenser System 14.1.4 Specimen and stage 14.1.5 The objective lens 14.1.6 The projector System 14.1.7 Viewing and recording 14.1.8 Protective measures: radiation, Vibration, magnetic
fields
168 168 169 170 170 170 171 173 174 174 175 175
177 177 178 179 179 179
180 181 181
184 184 184 185 186 187 187 189 190
192 192 192 196 199 201 203 204 204
206
Contents
14.2 Alternative operating modes 206 14.2.1 Hollow-cone brightfield 206 14.2.2 Darkfield 207 14.2.3 Electron spectroscopic imaging 208 14.2.4 The high-voltage electron microscope 210
Scanning microscopes 212 15.1 The specimen as source; basic features of scanning microscopy 212 15.2 Types of electron scanning microscopes 213 15.3 The SEM 214
15.3.1 The instrument 214 15.3.2 Factors affecting resolution 216 15.3.3 The secondary-electron mode 219 15.3.4 Backscattered emission modes 220
15.4 The STEM 221 15.4.1 Modes of STEM Operation 223
15.4.1.1 Elastic darkfield 223 15.4.1.2 Inelastic brightfield 224 15.4.1.3 Inelastic darkfield 224 15.4.1.4 X-ray mapping and energy-filtering modes 224 15.4.1.5 Ratio contrast and other processed modes 225
15.4.2 Analytical applications 225 15.4.2.1 Elemental analysis by quantitation of
electron energy loss 226 15.4.3 STEM applications in biology 226
Practical aspects of electron microscopy 228 16.1 Electron microscope specimens 228
16.1.1 Standard cellular preparations 228 16.1.2 Particulate materials 229 16.1.3 Low-temperature methods 231
16.1.3.1 Classical methods for preparing specimens at low temperatures 231
16.1.3.2 Cryomicroscopy 232 16.1.4 Specialized techniques 233
16.1.4.1 Autoradiography 233 16.1.4.2 Immunocytochemistry 234
16.1.5 Specimens for the SEM 234 16.1.5.1 Critical-point drying 234
16.2 Depths of field and focus and three-dimensional structure 235 16.2.1 Extent of field and focus 235 16.2.2 Deduction of three-dimensional structure 236
16.3 Determination of molecular weight 236 16.3.1 Mass mapping 237
16.4 Operation of the electron microscope 238 16.4.1 Operational procedures 238
16.4.1.1 Focusing 238
Contents XI
16.4.1.2 Compensation of astigmatism 240 16.4.1.3 Control of contamination 240 16.4.1.4 Alignment 241 16.4.1.5 Calibration 242 16.4.1.6 Resolution tests 244
16.4.2 Maintenance of image quality 244
17 The quest for ultimate electron microscopic resolution 247 17.1 Beam damage 247
17.1.1 Details of the problem 247 17.1.2 Possible and partial Solutions 248
17.2 Electron imaging 250 17.2.1 Summary of electron contrast mechanisms 250 17.2.2 Optimal contrast at resolutions down to about 2.0 nm 251 17.2.3 Contrast effects in direct imaging at high resolution 252 17.2.4 Selection of focal level 253
17.3 Image processing using Fourier analysis 253 17.3.1 The image-processing approach 254
17.3.1.1 The projection theorem 255 17.3.2 Techniques in image processing 256
17.3.2.1 Optical filtration 256 17.3.2.2 Computational methods 256 17.3.2.3 Retrieval of phases from the image 257
17.3.3 A specific application of image-processing methods in biology 259
17.4 The darkfield image 260 17.5 Ultimate resolution 264
17.5.1 A summary of limiting factors in electron microscopy 264 17.5.2 History and recent achievements 266 17.5.3 Prospects 267
18 Innovations in microscope development 270 18.1 The field-ion microscope 270 18.2 Ion microscopes 270 18.3 The neutron microscope 271 18.4 The photoelectron microscope 271 18.5 The scanning tunneling microscope and the atomic-force
microscope 271 18.6 Nuclear-magnetic-resonance microscopy 275 18.7 The acoustic microscope 276 18.8 "Superresolving" instruments and confocal Systems 278
18.8.1 The role of scanning in superresolution 279 18.8.2 Near-field microscopy 279 18.8.3 Comparison of confocal scanning microscope Systems 280 18.8.4 Operation of the tandem scanning mechanism in
confocal microscopy 282 18.9 Video-enhanced light microscope Systems 284
xii Contents
19 Photography 286 19.1 The nature of Photographie emulsions 286 19.2 Formation of the latent image 287 19.3 Chemical processing 288
19.3.1 Development 288 19.3.2 Fixation and washing 289
19.4 Properties of Photographie emulsions 291 19.4.1 Characteristic curves 291
19.4.1.1 Visiblelight 292 19.4.1.2 Electrons 293
19.4.2 Graininess and related quantities 294 19.4.3 Detective quantum efficiency 296
19.5 Basic features of color photography 296
Appendix: Image location 298 A Definitions 298 B Sign Conventions 300 C Equations that describe focusing by a Single surface 300 D Equations that describe focusing by a thin lens 300 E Equations that describe focusing by a thick lens 300 F Ray-tracing equations for exaet image location 301 G The sine theorem 301
Author index 303 Subject index 307
