Cellular Imaging Techniques for Neuroscience and Beyond

List of Contributors

1. Confocal Laser Scanning: of Instrument, Computer Processing, and Men

Introduction

Pinhole, Depth of Focus, and Laser Illumination

When/Why Does One Need a CLSM?

Abbe, Shannon, and Nyquist

Imaging of a 2D Line and Deblurring

Axial Resolution

Resolution and Sampling

Signal Separation, Orders of Magnitude, and Resolution Limits

Confocal Microscopy Further Considered

Cross Talk Awareness

Elimination of Cross Talk

Biological Objects Translated to Pixels

High-probability Determination of Diameter

Why Does a 3D Reconstructed Cell Resemble a Pancake?

Touch

Actual Experiment

Synaptic Contacts: Extra Marker

Colocalization

Conclusion

Acknowledgments

References

2. Beyond Abbe’s Resolution Barrier: Sted Microscopy

Introduction

A New Wave of Imaging

STED Microscopy: The Basic Concept

Implementation of STED Microscopy

Sine Qua Non: Speed, Color, Depth, Live Imaging

Summary and Outlook

References

3. Enhancement of Optical Resolution by 4pi Single and Multiphoton Confocal Fluorescence Microscopy

Introduction

The 4pi Principle and Setup

Microscope Alignment

4pi Imaging

4pi Deconvolution

Sample Preparation

Microtubule and Microtubule Plus End Imaging

Visualization of DNA

Single-photon Excitation (Measurement of the Redox State in Dopamine Neurons)

SYCP3 Axis as a Marker for Chromatin Organization in Mouse Spermatocytes

Microbubbles with Medicine

Future of 4pi Imaging

Acknowledgment

References

4. Nano Resolution Optical Imaging Through Localization Microscopy

Introduction

Superresolution Microscopy Techniques

The Main Approaches to Single-molecule Localization-based Superresolution Microscopy

Fluorescent Probes

Fluorescent Proteins

Multicolor Localization Microscopy

Outlook

Conclusion

Acknowledgments

References

5. Optical Investigation of Brain Networks Using Structured Illumination

Introduction

Structuring Light by Phase Modulation Using SLMs

Wavefront Engineering Using SLMs: The Optical Setup

Light-sensitive Molecular Tools for the Investigation of the Central Nervous System

SLM-based Approaches for the Optical Dissection of Brain Microcircuits

Conclusions

Acknowledgments

References

6. Multiphoton Microscopy Advances Toward Super Resolution

Introduction

Point Spread Function for Single- and Multiphoton Imaging

Super Resolution Techniques for Multiphoton Fluorescence Microscopy

Conclusions

Acknowledgments

Appendix

Transition Probability for Single-Photon Excitation

References

7. The Cell at Molecular Resolution: Principles and Applications of Cryo-Electron Tomography

Introduction: Cellular Landscapes at Molecular Resolution

The Cryo-ET Method

Detection, Identification, and Hybrid Methods

Conclusions

Acknowledgments

References

8. Cellular-Level Optical Biopsy Using Full-Field Optical Coherence Microscopy

Introduction

The FF-OCM Technique

Detection Sensitivity

Spatial Resolution

Sample Motion Artifacts

FF-OCM for High-Resolution “Optical Biopsy”

Conclusion

Acknowledgment

References

9. Retroviral Labeling and Imaging of Newborn Neurons in the Adult Brain

Techniques to Label and Detect Newborn Neurons in the Adult Brain

Retrovirus-mediated Labeling of Adult-born Neurons

Single-cell Genetic Manipulation in Adult-born Neurons

Retrovirus Production and Delivery

Viral-labeled Cell Toxicity and Physiological Changes

Imaging Newborn Neurons in the Adult Brain

In Vivo Live Animal Imaging of Adult Neurogenesis

In Vivo Window Preparation

In Vivo Imaging Setup and Acquisition

Postacquisition Image Processing and Analysis

Future Directions in Live Animal Imaging of Adult Neurogenesis

Acknowledgments

References

10. Study of Myelin Sheaths by Cars Microscopy

Traditional Myelin Imaging Methods

Principle and History of CARS Microscopy

Technical Characteristics of CARS Microscopy

CARS Microscopy for Ex Vivo and In Vivo Myelin Imaging

Mechanistic Understanding of Demyelination and Remyelination Enabled by CARS Imaging

Other Methods for In Vivo Imaging of Myelin

Outlook for Myelin Imaging by CARS Microscopy

Acknowledgments

References

11. High-Resolution Approaches to Studying Presynaptic Vesicle Dynamics Using Variants of Frap and Electron Microscopy

Introduction

Quantifying Dynamic Events at the Macromolecular Scale

FRAP for Studying Mobility

Variations on FRAP Using Photoswitchable Fluorophores

Linking Fluorescence and Ultrastructure: Correlative Approaches for Assaying Presynaptic Function

Structure–Function Relationships of Vesicle Pools in Hippocampal Synapses

Combining FRAP with Correlative Electron Microscope

Concluding Remarks

Acknowledgments

References

Index