Page 9 - Fundamentals of Light Microscopy and Electronic Imaging

viii CONTENTS

Formation of the DIC Image 159
Interference Between O and E Wavefronts
and the Application of Bias Retardation 160
Alignment of DIC Components 161
Image Interpretation 166
The Use of Compensators in DIC Microscopy 167
Comparison of DIC and Phase Contrast Optics 168
Modulation Contrast Microscopy 168
Contrast Methods Using Oblique Illumination 169
Alignment of the Modulation Contrast Microscope 172
Exercise: DIC Microscopy 173

11. FLUORESCENCE MICROSCOPY 177
Overview 177
Applications of Fluorescence Microscopy 178
Physical Basis of Fluorescence 179
Properties of Fluorescent Dyes 182
Demonstration: Fluorescence of Chlorophyll and Fluorescein 183
Autofluorescence of Endogenous Molecules 185
Demonstration: Fluorescence of Biological Materials
Under Ultraviolet Light 189
Arrangement of Filters and the Epi-illuminator
in the Fluorescence Microscope 189
Objective Lenses and Spatial Resolution in Fluorescence Microscopy 194
Causes of High-Fluorescence Background 196
The Problem of Bleed-Through with Multiply Stained Specimens 197
Examining Fluorescent Molecules in Living Cells 198
Exercise: Fluorescence Microscopy of Living Tissue Culture Cells 199
12. CONFOCAL LASER SCANNING MICROSCOPY 205
Overview 205
The Optical Principle of Confocal Imaging 208
Demonstration: Isolation of Focal Plane Signals
with a Confocal Pinhole 211
Advantages of CLSM Over Wide-Field Fluorescence Systems 213
Criteria Defining Image Quality and the Performance
of an Electronic Imaging System 215
Electronic Adjustments and Considerations
for Confocal Fluorescence Imaging 217
Photobleaching 223
General Procedure for Acquiring a Confocal Image 224
Two-Photon and Multi-Photon Laser Scanning Microscopy 226
Confocal Imaging with a Spinning Nipkow Disk 229
Exercise: Effect of Confocal Variables on Image Quality 230

13. VIDEO MICROSCOPY 233
Overview 233
Applications and Specimens Suitable for Video 233