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Concepts in Confocal Microscopy

Section Overview:

Laser scanning confocal microscopy represents one of the most significant advances in optical microscopy ever developed, primarily because the technique enables visualization deep within both living and fixed cells and tissues and affords the ability to collect sharply defined optical sections from which three-dimensional renderings can be created. The principles and techniques of confocal microscopy are becoming increasingly available to individual researchers as new single-laboratory microscopes are introduced. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.

Review Articles

  • Introduction to Confocal Microscopy

    Confocal microscopy has advantages over widefield optical microscopy, including the ability to eliminate or reduce background information away from the focal plane and collect serial optical sections from thick specimens.

  • Fluorophores for Confocal Microscopy

    Biological laser scanning confocal microscopy relies on fluorescence as an imaging mode due to the high degree of sensitivity afforded by the technique coupled with the ability to specifically target structural components.

  • NEW! - Laser Scanning Confocal Microscope Simulator

    Explore multi-laser fluorescence and differential interference contrast (DIC) confocal imaging using the Olympus FluoView FV1000 confocal microscope software interface as a model in this tutorial.

  • Fluorescence Excitation & Emission Fundamentals

    Fluorescence is a member of the luminescence family of processes in which molecules emit light from electronically excited states created by either a physical, mechanical (friction), or chemical mechanism.

  • Spectral Bleed-Through Artifacts in Confocal Microscopy

    Spectral bleed-through of fluorescence emission occurs due to the broad bandwidths and asymmetrical spectral profiles exhibited by fluorophores, can be a problem in laser scanning confocal fluorescence microscopy.

  • Choosing Fluorophore Combinations for Confocal Microscopy

    Explore the matching of dual fluorophores with efficient laser excitation lines and determination of the bleed-through level that can be expected as a function of the detection window wavelength profiles in this tutorial.

  • Interference Filters for Fluorescence Microscopy

    The utilization of specialized and advanced thin film, interference filters have enhanced the versatility of fluorescence techniques, far beyond the capabilities of the earlier use of gelatin and glass filters.

  • Non-Coherent Light Sources for Confocal Microscopy

    Reviewed in this article are the merits and limitations of non-coherent light sources in confocal microscopy, both as light sources for confocal illumination and as secondary sources for widefield microscopy in confocal microscopes.

  • Resolution & Contrast in Confocal Microscopy

    In a fluorescence microscope, contrast is determined by the number of photons collected, the dynamic range of the signal, optical aberrations of the imaging system, and the number of picture elements (pixels) per unit area.

  • Introduction to Lasers

    Lasers are designed to produce and amplify this stimulated form of light into intense and focused beams. The word laser was coined as an acronym for Light Amplification by the Stimulated Emission of Radiation.

  • Laser Systems for Confocal Microscopy

    The lasers used in laser scanning confocal microscopy are high-intensity monochromatic light sources, which are useful for techniques including optical trapping, lifetime imaging studies, and total internal reflection fluorescence.

  • Acousto-Optic Tunable Filters (AOTFs)

    Several benefits of the AOTF combine to enhance the versatility of the latest generation of confocal instruments, and these devices are becoming popular for control of excitation wavelength ranges and intensity.

  • Confocal Microscope Objectives

    The contrast and resolution of fine specimen detail, the depth within the specimen from which information can be obtained, and the lateral extent of the image field are all determined by the objective.

  • Confocal Microscope Scanning Systems

    Three principal scanning variations are employed to produce confocal microscope images. Each technique has features that make it advantageous for specific confocal applications, but limit the usefulness in others.

  • Signal-to-Noise Considerations

    In any quantitative assessment of imaging capabilities utilizing digital microscopy techniques, including confocal methods, the effect of signal sampling on contrast and resolution must be considered.

  • Electronic Light Detectors: Photomultipliers

    In laser scanning confocal microscopy, the collection of secondary emission gathered by the objective can be accomplished by classes of photosensitive detectors, including photomultipliers, photodiodes, and CCDs.

  • Electronic Imaging Detectors

    Over the past several years, the rapidly growing field of fluorescence microscopy has evolved from a dependence on traditional photomicrography using emulsion-based film to one in which electronic images are the output of choice.

  • Applications in Confocal Microscopy

    Applications available to laser scanning confocal microscopy includes a variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues.

  • Fluorochrome Data Table

    As a guide to fluorophores for confocal and widefield fluorescence microscopy, the table presented lists many commonly-used fluorochromes, with their respective peak absorption, and emission wavelengths.

  • Glossary of Terms in Confocal Microscopy

    Featured here are resources provided as a guide and reference tool for visitors who are exploring the large spectrum of specialized topics in fluorescence and laser scanning confocal microscopy.

  • Confocal Microscopy Interactive Tutorials

    Discover and explore the gallery of various interactive Java tutorials designed to explain and aid students visually in understanding complex and difficult concepts in confocal microscopy.

References and Resources

Confocal Microscopy Web Resources

Laser scanning confocal microscopy (LSCM) is a tool that has been extensively utilized for inspection of semiconductors, is now becoming a mainstream application in cell biology. The links provided in this section from the Olympus Microscopy Resource Center web site offer tutorials, instrumentation, application notes, technical support, glossaries, and reference materials on confocal microscopy and related techniques.

Contributing Authors

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, Tennessee, 37212.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Matthew Parry-Hill, Thomas J. Fellers, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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