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Related Experiment Videos

Confocal fluorescence microscopy with the tandem scanning light microscope.

S J Wright1, J S Walker, H Schatten

  • 1Integrated Microscopy Resource for Biomedical Research, University of Wisconsin, Madison 53706.

Journal of Cell Science
|December 1, 1989
PubMed
Summary

The tandem scanning confocal microscope (TSM) offers high-resolution 3D imaging of fluorescent biological structures. This advanced fluorescence microscopy technique minimizes photobleaching, enabling prolonged observation of living cells and detailed cellular component analysis.

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Area of Science:

  • Cell Biology
  • Microscopy
  • Biophysics

Background:

  • Confocal microscopy is crucial for high-resolution biological imaging.
  • Distinguishing 3D cellular structures requires advanced imaging techniques.
  • Traditional fluorescence microscopy faces limitations in resolution and photobleaching.

Purpose of the Study:

  • To describe the applications of the tandem scanning confocal microscope (TSM) in fluorescence microscopy.
  • To evaluate the TSM's capability in resolving fluorescent biological structures in three dimensions.
  • To demonstrate the TSM's utility for multi-fluorochrome labeling and prolonged live-cell imaging.

Main Methods:

  • Utilized a tandem scanning confocal microscope (TSM) coupled with a cooled charge-coupled device (cooled CCD).

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  • Employed conventional epifluorescence light sources and filter sets for excitation.
  • Acquired serial optical sections in the Z-axis for 3D reconstruction and stereo imaging.

    • Main Results:

    • Achieved high-resolution, confocal data distinguishing different fluorescent cellular components in 3D within single cells.
    • Successfully imaged fluorochromes excited by ultraviolet light (e.g., Hoechst, DAPI) alongside fluorescein and rhodamine.
    • Observed insignificant photobleaching due to dim illumination, allowing prolonged viewing of living specimens.

    Conclusions:

    • The TSM is a powerful tool for fluorescence microscopy, providing detailed 3D visualization of cellular structures.
    • Its ability to image multiple fluorochromes and its low photobleaching characteristics are advantageous for complex biological studies.
    • The TSM facilitates the determination of intricate three-dimensional relationships within cells labeled with various fluorescent markers.