Cellular Imaging Techniques for Neuroscience and Beyond

Cellular Imaging Techniques for Neuroscience and Beyond

1st Edition - August 8, 2012

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  • Editor: Floris G. Wouterlood
  • Hardcover ISBN: 9780123858726
  • eBook ISBN: 9780123858733

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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.

Key Features

  • Single and multiphoton confocal microscopy, and 4-pi confocal microscopy
  • Obtaining nanoresolution via photoactivation localization microscopy (PALM)
  • Several procedures that correlate observations in optical fluorescence microscopy and electron microscopy
  • Study of morphology and function via high-resolution fluorescence procedures
  • Additional high-resolution microscopic techniques


Researchers and graduate students in neuroscience; confocal "aficionados" in the cell biology community

Table of Contents

  • List of Contributors

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


    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?


    Actual Experiment

    Synaptic Contacts: Extra Marker





    2. Beyond Abbe’s Resolution Barrier: Sted Microscopy


    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


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


    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



    4. Nano Resolution Optical Imaging Through Localization Microscopy


    Superresolution Microscopy Techniques

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

    Fluorescent Probes

    Fluorescent Proteins

    Multicolor Localization Microscopy





    5. Optical Investigation of Brain Networks Using Structured Illumination


    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




    6. Multiphoton Microscopy Advances Toward Super Resolution


    Point Spread Function for Single- and Multiphoton Imaging

    Super Resolution Techniques for Multiphoton Fluorescence Microscopy




    Transition Probability for Single-Photon Excitation


    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




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


    The FF-OCM Technique

    Detection Sensitivity

    Spatial Resolution

    Sample Motion Artifacts

    FF-OCM for High-Resolution “Optical Biopsy”




    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



    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



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


    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




Product details

  • No. of pages: 296
  • Language: English
  • Copyright: © Academic Press 2012
  • Published: August 8, 2012
  • Imprint: Academic Press
  • Hardcover ISBN: 9780123858726
  • eBook ISBN: 9780123858733

About the Editor

Floris G. Wouterlood

Affiliations and Expertise

Associate Professor, Department of Anatomy & Neuroscience, Vrije University Medical Center, Amsterdam

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