List of Contributors ........................................... xi
Chapter 1 Confocal Laser Scanning: of Instrument, Computer
Processing, and Men ............................................. 1
Introduction .................................................... 2
Pinhole, Depth of Focus, and Laser Illumination ................. 2
When/Why Does One Need a CLSM? .................................. 4
Abbe, Shannon, and Nyquist ...................................... 8
Imaging of a 2D Line and Deblurring ............................. 9
Axial Resolution ............................................... 11
Resolution and Sampling ........................................ 12
Signal Separation, Orders of Magnitude, and Resolution Limits .. 13
Confocal Microscopy Further Considered ......................... 14
Cross Talk Awareness ........................................... 16
Elimination of Cross Talk ...................................... 18
Biological Objects Translated to Pixels ........................ 19
High-probability Determination of Diameter ..................... 20
Why Does a 3D Reconstructed Cell Resemble a Pancake? ........... 24
Touch .......................................................... 25
Actual Experiment .............................................. 26
Synaptic Contacts: Extra Marker ................................ 29
Colocalization ................................................. 30
Conclusion ..................................................... 32
Acknowledgments ................................................ 32
References ..................................................... 33
Chapter 2 Beyond Abbe's Resolution Barrier: STED Microscopy ... 35
Introduction ................................................... 36
A New Wave of Imaging .......................................... 37
STED Microscopy: The Basic Concept ............................. 39
Implementation of STED Microscopy .............................. 42
Sine Qua Non: Speed, Color, Depth, Live Imaging ................ 47
Summary and Outlook ............................................ 52
References ..................................................... 52
Chapter 3 Enhancement of Optical Resolution by 4pi Single
and Multiphoton Confocal Fluorescence Microscopy ............... 55
Introduction ................................................... 56
The 4pi Principle and Setup .................................... 56
Microscope Alignment ........................................... 58
4pi Imaging .................................................... 60
4pi Deconvolution .............................................. 61
Sample Preparation ............................................. 62
Microtubule and Microtubule Plus End Imaging ................... 65
Visualization of DNA ........................................... 66
Single-photon Excitation (Measurement of the Redox State in
Dopamine Neurons) .............................................. 68
SYCP3 Axis as a Marker for Chromatin Organization in Mouse
Spermatocytes ............................................... 70
Microbubbles with Medicine ..................................... 75
Future of 4pi Imaging .......................................... 77
Acknowledgment ................................................. 77
References ..................................................... 77
Chapter 4 Nano Resolution Optical Imaging Through
Localization Microscopy ........................................ 81
Introduction ................................................... 82
Superresolution Microscopy Techniques .......................... 82
The Main Approaches to Single-molecule Localization-based
Superresolution Microscopy .................................. 87
Fluorescent Probes ............................................. 89
Fluorescent Proteins ........................................... 89
Multicolor Localization Microscopy ............................. 90
Outlook ........................................................ 93
Conclusion ..................................................... 96
Acknowledgments ................................................ 96
References ..................................................... 97
Chapter 5 Optical Investigation of Brain Networks Using
Structured Illumination ....................................... 101
Introduction .................................................. 101
Structuring Light by Phase Modulation Using SLMs .............. 103
Wavefront Engineering Using SLMs: The Optical Setup ........... 104
Light-sensitive MolecularTools for the Investigation of the
Central Nervous System ..................................... 111
SLM-based Approaches for the Optical Dissection of Brain
Microcircuits .............................................. 112
Conclusions ................................................... 116
Acknowledgments ............................................... 116
References .................................................... 116
Chapter 6 Multiphoton Microscopy Advances Toward
Super Resolution .............................................. 121
Introduction .................................................. 121
Point Spread Function for Single- and Multiphoton Imaging ..... 123
Super ResolutionTechniques for Multiphoton Fluorescence
Microscopy ................................................. 126
Conclusions ................................................... 134
Acknowledgments ............................................... 134
References .................................................... 134
Chapter 7 The Cell at Molecular Resolution: Principles
and Applications of Cryo-Electron Tomography .................. 141
Introduction: Cellular Landscapes at Molecular Resolution ..... 141
The Cryo-ET Method ............................................ 143
Detection, Identification, and Hybrid Methods ................. 151
Conclusions ................................................... 175
Acknowledgments ............................................... 178
References .................................................... 178
Chapter 8 Cellular-Level Optical Biopsy Using Full-Field
Optical Coherence Microscopy .................................. 185
Introduction .................................................. 185
The FF-OCMTechnique ........................................... 187
Detection Sensitivity ......................................... 189
Spatial Resolution ............................................ 191
Sample Motion Artifacts ....................................... 192
FF-OCM for High-Resolution "Optical Biopsy" ................... 193
Conclusion .................................................... 195
Acknowledgment ................................................ 196
References .................................................... 196
Chapter 9 Retroviral Labeling and Imaging of Newborn
Neurons in the Adult Brain .................................... 201
Techniques to Label and Detect Newborn Neurons in the Adult
Brain ......................................................... 202
Retrovirus-mediated Labeling of Adult-born Neurons ............ 203
Single-cell Genetic Manipulation in Adult-born Neurons ........ 205
Retrovirus Production and Delivery ............................ 207
Viral-labeled Cell Toxicity and Physiological Changes ......... 209
Imaging Newborn Neurons in the Adult Brain .................... 210
In Vivo Live Animal Imaging of Adult Neurogenesis ............. 210
In Vivo Window Preparation .................................... 211
In Vivo Imaging Setup and Acquisition ......................... 213
Postacquisition Image Processing and Analysis ................. 213
Future Directions in Live Animal Imaging of Adult
Neurogenesis ............................................... 214
Acknowledgments ............................................... 216
References .................................................... 216
Chapter 10 Study of Myelin Sheaths by CARS Microscopy ........ 221
Traditional Myelin Imaging Methods ............................ 221
Principle and History of CARS Microscopy ...................... 224
Technical Characteristics of CARS Microscopy .................. 226
CARS Microscopy for Ex Vivo and In Vivo Myelin Imaging ........ 227
Mechanistic Understanding of Demyelination and Remyelination
Enabled by CARS Imaging .................................... 231
Other Methods for In Vivo Imaging of Myelin ................... 236
Outlook for Myelin Imaging by CARS Microscopy ................. 238
Acknowledgments ............................................... 240
References .................................................... 240
Chapter 11 High-Resolution Approaches to Studying
Presynaptic Vesicle Dynamics Using Variants of FRAP and
Electron Microscopy ........................................... 247
Introduction .................................................. 248
Quantifying Dynamic Events at the Macromolecular Scale ........ 249
FRAP for Studying Mobility .................................... 251
Variations on FRAP Using Photoswitchable Fluorophores ......... 255
Linking Fluorescence and Ultrastructure: Correlative
Approaches for Assaying Presynaptic Function ............... 259
Structure-Function Relationships of Vesicle Pools in
Hippocampal Synapses ....................................... 263
Combining FRAP with Correlative Electron Microscope ........... 265
Concluding Remarks ............................................ 269
Acknowledgments ............................................... 269
References .................................................... 270
Index ......................................................... 275
Color Plates
|
