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Effect of probe diffusion on the SOFI imaging accuracy.

Wim Vandenberg1, Peter Dedecker1

  • 1Department of Chemistry, KU Leuven, Celestijnenlaan 200G, 3001 Heverlee, Belgium.

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Probe diffusion in live-cell super-resolution optical fluctuation imaging (SOFI) can alter signal amplitude but enhances imaging quality by preventing undersampling. This effect is minimal for photochromic fluorescent proteins in practical biological experiments.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Live-cell super-resolution fluorescence imaging is crucial for biological research.
  • Sample dynamics, such as probe diffusion, can impact imaging quality.
  • Super-resolution optical fluctuation imaging (SOFI) is a key technique.

Purpose of the Study:

  • To evaluate the effect of probe diffusion on SOFI.
  • To analyze how fluorophore diffusion influences SOFI signal amplitude and image quality.
  • To assess the impact of diffusion on live cells labeled with photochromic fluorescent proteins.

Main Methods:

  • Development of a theoretical model for SOFI.
  • Numerical simulations of live-cell imaging.
  • Utilizing photochromic fluorescent proteins for labeling.

Main Results:

  • Fluorophore diffusion alters the amplitude of the SOFI signal under physiological conditions.
  • The change in amplitude is proportional to the fluorophore on-time ratio.
  • Probe diffusion enhances SOFI by mitigating spatial undersampling due to limited labeling density.

Conclusions:

  • Probe diffusion does not significantly distort SOFI in practical biological experiments with photochromic fluorescent proteins.
  • Diffusion is beneficial for SOFI, improving resolution by overcoming labeling density limitations.
  • SOFI remains a robust technique for live-cell imaging despite probe dynamics.