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Related Experiment Videos

Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy.

S E D Webb1, S R Needham, S K Roberts

  • 1CCLRC Daresbury Laboratory, Warrington, UK. stephen.webb@dl.ac.uk

Optics Letters
|June 24, 2006
PubMed
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We developed a new microscope for imaging single molecules in live cells using wavelength and polarization. This technique reveals energy transfer between molecules on cell membranes, highlighting the importance of polarization discrimination.

Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Single-molecule imaging is crucial for understanding cellular processes.
  • Current microscopy techniques have limitations in resolving molecular interactions in live cells.

Purpose of the Study:

  • To develop and demonstrate a wide-field total-internal-reflection fluorescence microscope for live-cell imaging.
  • To visualize single-molecule fluorescence resonance energy transfer (FRET) between molecules in the plasma membrane.
  • To emphasize the significance of polarization discrimination in addition to wavelength separation for molecular analysis.

Main Methods:

  • Development of a wide-field total-internal-reflection fluorescence microscope.
  • Imaging single molecules in live cells with resolution in both wavelength and polarization.

Related Experiment Videos

  • Measuring fluorescence resonance energy transfer (FRET) between single pairs of fluorescent molecules.

    • Main Results:

    • Successfully imaged single molecules in live cells with high resolution.
    • Observed FRET between single fluorescent molecules bound to signaling receptors in the plasma membrane.
    • Demonstrated that polarization discrimination is essential for accurate molecular analysis, alongside wavelength separation.

    Conclusions:

    • The developed microscope enables advanced live-cell imaging of single molecules.
    • This technique provides new insights into molecular interactions, such as FRET, at the cell membrane.
    • Polarization discrimination is a critical parameter for resolving complex molecular events in biological systems.