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Local dimensionality determines imaging speed in localization microscopy.

Patrick Fox-Roberts1, Richard Marsh1, Karin Pfisterer1

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Live-cell super-resolution microscopy speed depends on sample structure. Exceeding optimal fluorophore density causes silent failure and artifacts, necessitating new analysis tools for accurate imaging.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Localization microscopy achieves nanoscale resolution, but live-cell super-resolution imaging is limited by speed.
  • Current methods often assume fixed imaging speeds, neglecting sample-specific dynamics.

Purpose of the Study:

  • To investigate the impact of sample dimensionality on achievable imaging speed in localization microscopy.
  • To identify optimal fluorophore activation densities for different biological structures.
  • To develop a tool for detecting and correcting analysis failures due to high activation density.

Main Methods:

  • Simulated and experimental analysis of localization microscopy data across varying sample dimensions (point, strand, extended structures).
  • Systematic variation of fluorophore activation density and excitation power.
  • Development and validation of a computational tool for artifact detection.

Main Results:

  • Imaging speed is highly dependent on sample dimensionality, varying by orders of magnitude.
  • Excessive fluorophore activation density leads to silent failure and reconstruction artifacts.
  • The developed tool successfully identifies and allows correction for high-density-induced artifacts.

Conclusions:

  • Optimizing fluorophore density is crucial for high-speed live-cell super-resolution imaging.
  • Sample dimensionality is a key factor influencing maximum imaging speed.
  • The released tool enhances the reliability of localization microscopy for both live and fixed cells.