| Preface |
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I. Considerations for Routine Imaging |
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Entering the Portal: Understanding the Digital Image Recorded Through a Microscope |
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3 | (42) |
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3 | (1) |
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4 | (1) |
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Digital Image Acquisition: Analog to Digital Conversion |
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4 | (2) |
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Spatial Resolution in Digital Images |
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6 | (2) |
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The Contrast Transfer Function |
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8 | (2) |
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Image Brightness and Bit Depth |
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10 | (1) |
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11 | (1) |
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Fundamental Properties of CCD Cameras |
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12 | (4) |
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CCD Enhancing Technologies |
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16 | (1) |
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17 | (4) |
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21 | (3) |
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The Point-Spread Function |
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24 | (4) |
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Digital Image Display and Storage |
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28 | (1) |
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Imaging Modes in Optical Microscopy |
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29 | (10) |
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39 | (2) |
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41 | (4) |
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41 | (4) |
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Quantitative Biological Image Analysis |
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45 | (26) |
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45 | (1) |
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Definitions and Perspectives |
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46 | (2) |
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48 | (9) |
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Image Intensity Transformation |
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50 | (1) |
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50 | (3) |
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Geometrical Image Transformation |
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53 | (2) |
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55 | (2) |
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Advanced Processing for Image Analysis |
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57 | (6) |
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58 | (1) |
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Neuron Tracing and Quantification |
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58 | (2) |
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Particle Detection and Tracking |
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60 | (2) |
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Cell Segmentation and Tracking |
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62 | (1) |
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Higher-Dimensional Data Visualization |
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63 | (3) |
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64 | (1) |
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64 | (2) |
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Software Tools and Development |
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66 | (5) |
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68 | (3) |
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The Open Microscopy Environment: A Collaborative Data Modeling and Software Development Project for Biological Image Informatics |
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71 | (22) |
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71 | (3) |
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72 | (1) |
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Why OME - What Is the Problem? |
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72 | (2) |
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OME Specifications and File Formats |
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74 | (3) |
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74 | (2) |
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OME-XML, OME-TIFF and Bio-Formats |
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76 | (1) |
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OME Data Management and Analysis Software |
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77 | (13) |
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OME Server and Web User Interface |
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77 | (7) |
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OMERO Server, Client and Importer |
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84 | (5) |
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Developing Usable Tools for Imaging |
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89 | (1) |
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Conclusions and Future Directions |
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90 | (3) |
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90 | (3) |
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Design and Function of a Light-Microscopy Facility |
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93 | (24) |
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93 | (2) |
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95 | (1) |
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96 | (2) |
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96 | (1) |
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97 | (1) |
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97 | (1) |
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98 | (5) |
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98 | (1) |
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99 | (1) |
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100 | (1) |
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100 | (3) |
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103 | (9) |
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Equipment-Booking Database |
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103 | (3) |
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106 | (1) |
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107 | (4) |
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111 | (1) |
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112 | (5) |
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113 | (4) |
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II. Advanced Methods and Concepts |
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Quantitative Colocalisation Imaging: Concepts, Measurements, and Pitfalls |
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117 | (40) |
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117 | (20) |
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One Fluorophore, One Image? |
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124 | (11) |
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A Practical Example of Dual-Band Detection |
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135 | (2) |
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Quantifying Colocalisation |
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137 | (13) |
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137 | (2) |
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139 | (8) |
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147 | (3) |
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150 | (7) |
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151 | (6) |
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Quantitative FRET Microscopy of Live Cells |
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157 | (26) |
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157 | (1) |
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Introductory Physics of FRET |
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158 | (2) |
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Manifestations of FRET in Fluorescence Signals |
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160 | (3) |
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Spectral Change (Sensitized Emission) |
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160 | (1) |
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161 | (1) |
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162 | (1) |
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Accelerated Photobleaching |
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162 | (1) |
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Molecular Interaction Mechanisms That Can Be Observed by FRET |
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163 | (2) |
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164 | (1) |
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164 | (1) |
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164 | (1) |
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Measuring Fluorescence Signals in the Microscope |
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165 | (2) |
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Methods for FRET Microscopy |
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167 | (8) |
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Photobleaching Approaches |
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168 | (2) |
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170 | (3) |
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Spectral Fingerprinting and Matrix Notation for FRET |
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173 | (1) |
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174 | (1) |
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Fluorescence Lifetime Imaging Microscopy for FRET |
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175 | (1) |
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Data Display and Interpretation |
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176 | (1) |
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177 | (1) |
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FRET Microscopy for Analyzing Interaction Networks in Live Cells |
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178 | (2) |
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180 | (3) |
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180 | (3) |
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Fluorescence Photobleaching and Fluorescence Correlation Spectroscopy: Two Complementary Technologies To Study Molecular Dynamics in Living Cells |
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183 | (52) |
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183 | (6) |
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FRAP and Other Photobleaching Methods |
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184 | (2) |
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FCS and Other Fluctuation Analysis Methods |
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186 | (1) |
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Comparing and Combining Techniques |
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187 | (2) |
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189 | (7) |
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189 | (2) |
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191 | (2) |
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Diffusion and Binding in Living Cells |
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193 | (1) |
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Fluorescence, Blinking, and Photobleaching |
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194 | (1) |
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195 | (1) |
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How To Perform a FRAP Experiment |
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196 | (9) |
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The Principle of Imaging-Based FRAP |
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196 | (1) |
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Choosing and Optimising the Experimental Parameters |
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197 | (3) |
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200 | (3) |
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Controls and Potential Artefacts |
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203 | (2) |
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How To Perform an FCS Experiment |
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205 | (12) |
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205 | (3) |
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Instrument Alignment and Calibration |
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208 | (4) |
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212 | (1) |
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213 | (2) |
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215 | (2) |
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How To Perform a CP Experiment |
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217 | (4) |
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217 | (1) |
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Choosing and Optimising the Experimental Parameters |
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218 | (1) |
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219 | (1) |
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Controls and Potential Artefacts |
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220 | (1) |
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221 | (6) |
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Fluorescence Recovery After Photobleaching |
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221 | (2) |
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Fluorescence Correlation Spectroscopy |
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223 | (3) |
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Continuous Fluorescence Photobleaching |
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226 | (1) |
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227 | (8) |
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227 | (8) |
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Single Fluorescent Molecule Tracking in Live Cells |
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235 | (30) |
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235 | (1) |
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Tracking of Single Chromosomal Loci |
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236 | (11) |
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236 | (1) |
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In Vivo Single Loci Tagging via Operator/Represser Recognition |
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237 | (1) |
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The Design of Strains Containing TetO Repeats and Expressing TetR-GFP |
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238 | (6) |
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In Vivo Microscopy for Visualization of Single Tagged Chromosomal Loci |
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244 | (2) |
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Limits and Extension of Operator/Represser Single Loci Tagging System |
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246 | (1) |
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Single-Molecule Tracking of mRNA |
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247 | (6) |
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247 | (1) |
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247 | (1) |
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The Molecular Beacon System |
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248 | (2) |
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Setting Up the Molecular Beacon System for the Detection of mRNA |
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250 | (1) |
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Ensuring the Observed Fluorescent Particles in Vivo Consist of Single Molecules of mRNA |
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251 | (2) |
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Single-Particle Tracking for Membrane Proteins |
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253 | (5) |
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253 | (1) |
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Quantum Dots As Fluorescent Labels for Biological Samples |
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254 | (1) |
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Functionalizing Quantum Dots To Label Specific Proteins |
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255 | (2) |
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Tracking the Glycin Receptor 1 at the Synaptic Cleft Using Quantum Dots |
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257 | (1) |
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Tracking Analysis and Image Processing of Data from Particle Tracking in Living Cells |
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258 | (1) |
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258 | (1) |
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Protocols for Laboratory Use |
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259 | (6) |
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Protocol: Single-Molecule Tracking of Chromosomal Loci in Yeast |
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259 | (1) |
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Protocol: Single-Molecule Tracking of mRNA - Experiment Using Molecular Beacons |
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259 | (2) |
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261 | (4) |
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From Live-Cell Microscopy to Molecular Mechanisms: Deciphering the Functions of Kinetochore Proteins |
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265 | (24) |
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265 | (3) |
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Biological Problem: Deciphering the Functions of Kinetochore Proteins |
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268 | (1) |
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269 | (4) |
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Extraction of Dynamics from Images |
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273 | (3) |
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274 | (1) |
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275 | (1) |
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275 | (1) |
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Characterization of Dynamics |
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276 | (6) |
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Confined Brownian Motion Model |
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277 | (1) |
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Simple Microtubule Dynamic Instability Model |
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278 | (1) |
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Autoregressive Moving Average Model |
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279 | (1) |
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Descriptor Sensitivity and Completeness |
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280 | (2) |
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Quantitative Genetics of the Yeast Kinetochore |
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282 | (2) |
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284 | (5) |
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284 | (5) |
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III. Cutting Edge Applications & Utilities |
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Towards Imaging the Dynamics of Protein Signalling |
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289 | (24) |
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Spatiotemporal Aspects of Protein Signalling Dynamics |
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289 | (1) |
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How To Be Fast While Maintaining the Resolution |
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290 | (9) |
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How To Make Proteins Visible |
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299 | (4) |
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Concepts To Image Protein Dynamics |
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303 | (2) |
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Concepts To Image Protein-Protein Interactions |
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305 | (4) |
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Concepts To Image Biochemistry with Fluorescent Proteins |
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309 | (4) |
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311 | (2) |
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New Technologies for Imaging and Analysis of Individual Microbial Cells |
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313 | (32) |
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313 | (1) |
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314 | (1) |
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315 | (3) |
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Imaging Single Molecules (Within Single Cells) |
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318 | (1) |
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Measuring Discrete Cell Properties and Processes |
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319 | (2) |
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321 | (2) |
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Hardware and Applications |
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323 | (3) |
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323 | (1) |
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324 | (1) |
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325 | (1) |
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Fluorescence Correlation Spectroscopy |
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326 | (4) |
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A Picture is Worth a Thousand Dots - New Developments in Flow Cytometry |
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330 | (4) |
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Strength in Numbers - Highly Parallel Analysis Using Cellular Arrays |
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334 | (1) |
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Nontactile Manipulation of Individual Cells and ``Wall-less Test Tubes'' |
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335 | (2) |
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337 | (8) |
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338 | (7) |
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Imaging Parasites in Vivo |
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345 | (20) |
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345 | (1) |
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The Life Cycle of Malaria Parasites |
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346 | (2) |
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A Very Brief History of Light Microscopy and Malaria Parasites |
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348 | (1) |
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In Vivo Imaging of Luminescent Parasites |
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349 | (1) |
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In Vivo Imaging of Fluorescent Parasites |
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350 | (1) |
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Imaging Malaria Parasites in the Mosquito |
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351 | (3) |
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Imaging Malaria Parasites in the Mammalian Host |
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354 | (4) |
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Towards Molecular Imaging in Vivo |
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358 | (1) |
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A Look at Other Parasites |
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359 | (1) |
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360 | (5) |
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360 | (5) |
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Computer-Assisted Systems for Dynamic 3D Reconstruction and Motion Analysis of Living Cells |
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365 | (20) |
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365 | (1) |
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Approaches to 3D Reconstruction and Motion Analysis |
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366 | (2) |
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Obtaining Optical Sections for 3D Reconstruction |
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368 | (1) |
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368 | (5) |
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Reconstructing 3D Faceted Images and Internal Architecture |
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373 | (1) |
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Quantitative Analyses of Behavior |
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373 | (2) |
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375 | (2) |
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377 | (3) |
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The Combined Use of LSCM and 3D-DIAS |
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380 | (1) |
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Reasons for 3D Dynamic Image Reconstruction Analysis |
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381 | (4) |
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382 | (3) |
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High-Throughput/High-Content Automated Image Acquisition and Analysis |
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385 | (22) |
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The Driving Forces for High-Throughput/High-Content Automated Imaging |
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385 | (1) |
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Confocal Imaging in High Throughput - The Principles Available |
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386 | (3) |
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Resolution and Sensitivity |
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389 | (3) |
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392 | (1) |
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Where Is the Signal and How To Focus? |
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393 | (1) |
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394 | (1) |
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395 | (4) |
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Throughput: How To Acquire and Analyze Data Rapidly |
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399 | (2) |
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401 | (6) |
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404 | (3) |
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Cognition Network Technology - A Novel Multimodal Image Analysis Technique for Automatic Identification and Quantification of Biological Image Contents |
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407 | (16) |
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407 | (2) |
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Cognition Network Technology and Cognition Network Language |
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409 | (12) |
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409 | (1) |
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Input Data and Image Object Hierarchy |
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410 | (1) |
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411 | (2) |
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Classes and Classification |
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413 | (1) |
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414 | (1) |
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414 | (1) |
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Using CNT-CNL for Image Analysis |
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415 | (2) |
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417 | (4) |
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421 | (2) |
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421 | (2) |
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High-Content Phenotypic Cell-Based Assays |
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423 | (20) |
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A New Tool for Biological Research and Drug Discovery |
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423 | (1) |
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What Is High-Content Screening and How Can Biologists Use It? |
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424 | (1) |
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Assay Design: First Think, Then Act |
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425 | (1) |
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426 | (1) |
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426 | (2) |
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428 | (1) |
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428 | (2) |
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430 | (1) |
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431 | (1) |
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Transfection Optimization for RNAi-Based Assays |
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431 | (1) |
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Escapers and Silencing Efficiency |
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432 | (3) |
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435 | (1) |
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Off-Target or Unspecific Reactions |
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436 | (1) |
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437 | (1) |
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438 | (2) |
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440 | (3) |
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440 | (3) |
| Index |
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443 | |
