Description

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

Readership

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

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 Su

Details

No. of pages:
296
Language:
English
Copyright:
© 2012
Published:
Imprint:
Academic Press
Electronic ISBN:
9780123858733
Print ISBN:
9780123858726

About the editor

Reviews

"Wouterlood…introduces the confocal principle which eliminates out-of-focus haze, its components, and relevant equations. International scientists explain the principles and related methods of stimulated emission depletion (SRED), single molecule localization, and coherent anti-Stokes Raman (CARS) microscopy; labeling approaches; preparation of samples for imaging; and applications of, and developments in, this new wave of imaging, e.g., visualization of neuronal networks, DNA, and myelin." --Reference and Research Book News, February 2013