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Applications in Confocal Microscopy

Section Overview:

The broad range of applications available to laser scanning confocal microscopy includes a wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues. In addition, the growing use of new fluorescent proteins is rapidly expanding the number of original research reports coupling these useful tools to modern microscopic investigations. Other applications include resonance energy transfer, stem cell research, photobleaching studies, lifetime imaging, multiphoton microscopy, total internal reflection, DNA hybridization, membrane and ion probes, bioluminescent proteins, and epitope tagging. Many of these powerful techniques are described in the sections listed below.

Review Articles

  • Colocalization of Fluorophores in Confocal Microscopy

    During the digital recording of labeled fluorescent specimens, two or more of the emission signals can overlap in the final image due to their close proximity within the microscopic structure, this is known as colocalization.

  • Classification and Applications of Fluorescent Probes

    The tabulation in this section reviews examples of probes in each of the important biological classes, including nucleic acids, polysaccharides, lipids, membranes, cytoplasm, ion concentration, and specific organelles.

  • Chameleons: Calcium Ion Probes

    Chameleons are a new class of indicators for calcium ion concentrations in living cells, which operate through a conformational change that results in fluorescence resonance energy transfer in the presence of calcium ions.

  • Embryonic Stem Cells

    Embryonic stem cell lines, which were originally produced from the inner core of human blastocysts as well as those of other mammals, are now widely established in the research community using traditional in vitro culture.

  • Epitope Tagging

    An epitope (also known as an antigenic determinant) is a biological structure or sequence, such as a protein or carbohydrate, which is recognized by an antibody as an antigen.

  • Fiber FISH (Fluorescence in situ Hybridization)

    The term Fiber FISH (acronym for Fluorescence in situHybridization) refers to the common practice of fluorescence in situ (FISH) conducted on preparations of extended chromatin fibers.

  • Fluorescence Lifetime Imaging Microscopy (FLIM)

    Discussions reviewed in this section involve several important aspects of fluorescence lifetime imaging microscopy (FLIM), a new fluorescence microscopy technology.

  • Fluorescence Resonance Energy Transfer (FRET) Microscopy

    The technique of FRET, when applied to optical microscopy, permits determination of the approach between two molecules within several nanometers, a distance sufficiently close for molecular interactions to occur.

  • Fluorescence Photobleaching Investigations

    Both fluorescence loss in photobleaching (FLIP) and the recovery after photobleaching (FRAP) are techniques for observing the movement of intracellular materials through photobleaching of fluorescence.

  • Fluorescent and Bioluminescent Proteins

    Fluorescent proteins are widely used in research applications. Spectral variants of these fluorescent proteins are commercially available, and open up the possibility of multiple labeling experiments in living cells.

  • Green Fluorescent Protein (GFP)

    The green fluorescent protein (GFP) and its spectral variants (yellow, YFP); cyan, (CFP); blue, (BFP); and red (dsRFP) are rapidly becoming important investigational tools in the disciplines associated with the life sciences.

  • The Fluorescent Protein Color Palette

    A broad range of fluorescent protein genetic variants have been developed over the past several years that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum.

  • Optical Highlighter Fluorescent Proteins

    The discovery of fluorescent proteins and refinements in the genetic properties of these probes to generate an array of emission bandwidth profiles has helped biologists to visualize and track molecular events in cells.

  • Multiphoton Microscopy

    Multiphoton microscopy combines the optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture images of specimens tagged with specific fluorophores.

  • Fluorescent Protein Literature Sources

    The references listed in the following sections contain links to indexing services in order to provide investigators with a quick pathway to the original literature targeting fluorescent proteins.

  • Total Internal Reflection Fluorescence Microscopy

    Total internal reflection fluorescence microscopy (TIRFM) is an elegant optical technique utilized to observe single molecule fluorescence at surfaces and interfaces.

  • Specimen Preparation Using Synthetic Fluorophores and Indirect Immunofluorescence

    Listed are specimen preparation techniques using synthetic fluorophores coupled to immunofluorescence needed to investigate fixed adherent cells.

Interactive Java Tutorials

Colocalization of Fluorophores in Confocal Microscopy

Two or more fluorescence emission signals can often overlap in digital images recorded by confocal microscopy due to their close proximity within the specimen. This effect is known as colocalization and usually occurs when fluorescently labeled molecules bind to targets that lie in very close or identical spatial positions. This interactive tutorial explores the quantitative analysis of colocalization in a wide spectrum of specimens that were specifically designed either to demonstrate the phenomenon, or to alternatively provide examples of fluorophore targets that lack any significant degree of colocalization.

Contributing Authors

Will Casavan and Yuri Gaidoukevitch - Media Cybernetics, 8484 Georgia Avenue, Suite 200, Silver Spring, Maryland, 20910.

Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

Matthew J. Parry-Hill, Nathan S. Claxton, 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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