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The imaging of small cellular components requires powerful instruments, and an entire family of equipment and techniques based on the confocal principle has been developed over th… Read more
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The imaging of small cellular components requires powerful instruments, and an entire family of equipment and techniques based on the confocal principle has been developed over the past 30 years. Such methods are commonly used by neuroscience researchers, but the majority of these users do not have a microscopy or a cell biology backgrounds and do can encounter difficulties in obtaining and interpreting results. This volume brings experts in high-resolution optical microscopy applications in neuroscience and cell biology together to document the state of the art. Outlining what is currently possible, the volume also discusses promising developments for the future and aids readers in selecting the most scientifically meaningful approach to solve their questions. Each chapter discusses instrumentation and technology in relationship to application in research. All of the common and cutting edge trends are covered - fluorescence / laser electron / nonlinear microscopy, infrared fluorescence, multiphoton imaging, tomography, FRAP, live imaging, STED, PALM/STORM, etc.
Researchers and graduate students in neuroscience; confocal "aficionados" in the cell biology community
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
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