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Related Experiment Videos

Fluorescence photobleaching recovery using total internal reflection interference fringes.

Guy M Hagen1, Deborah A Roess, B George Barisas

  • 1Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA.

Analytical Biochemistry
|August 1, 2006
PubMed
Summary

This study introduces a new total internal reflection interference fringe fluorescence photobleaching recovery (FPR) method. It accurately measures cell membrane molecule dynamics by exclusively exciting surface fluorescence, overcoming intracellular interference.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Fluorescence photobleaching recovery (FPR) is crucial for studying cell membrane molecule dynamics.
  • Conventional FPR methods struggle with intracellular fluorescence interference, particularly with visible fluorescent protein (VFP) fusion proteins.
  • Accurate measurements of membrane molecule diffusion, size, and interactions are essential.

Purpose of the Study:

  • To develop and validate a novel FPR technique to eliminate intracellular fluorescence interference.
  • To accurately measure the lateral dynamics of cell membrane molecules, including VFP fusion proteins.
  • To improve the reliability and scope of FPR measurements for cell surface studies.

Main Methods:

  • Utilizing total internal reflection interference fringe FPR with a high numerical aperture (NA) objective.

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  • Employing interfering laser beams to create an extended interference pattern at the coverslip-medium interface.
  • Exciting fluorescence exclusively from molecules at the cell-coverslip contact surface.

    • Main Results:

    • The new method successfully rejects interfering intracellular fluorescence.
    • Accurate measurements of membrane dynamics were achieved for both VFP-containing and conventionally labeled molecules.
    • The technique interrogates a larger area, yielding more diffusion information per measurement compared to spot methods.

    Conclusions:

    • Total internal reflection interference fringe FPR is a robust technique for precise cell membrane dynamics studies.
    • This method significantly enhances the accuracy of FPR by isolating surface fluorescence.
    • It offers a superior alternative for studying membrane proteins and their interactions.