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Simultaneously Measuring Image Features and Resolution in Live-Cell STED Images.

Andrew E S Barentine1, Lena K Schroeder2, Michael Graff2

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Accurately measuring microscope resolution is vital for cell imaging. A new method, nested-loop ensemble PSF fitting, measures resolution in situ using cellular structures, eliminating the need for beads.

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Area of Science:

  • Microscopy
  • Cell Biology
  • Biophysics

Background:

  • Accurate estimation of microscope resolution is crucial for quantifying cellular features in fluorescence microscopy.
  • Traditional bead-based methods are insufficient for advanced techniques like stimulated emission depletion (STED) microscopy, where resolution is fluorophore- and parameter-dependent.

Purpose of the Study:

  • To develop a novel in situ method for measuring microscope resolution using known cellular geometries.
  • To overcome limitations of bead-based calibration in super-resolution microscopy.

Main Methods:

  • Developed nested-loop ensemble point spread function (PSF) fitting to decouple tubule diameter from PSF width.
  • Applied the method to analyze fixed-cell and live-cell images without precalibration or beads.

Main Results:

  • The nested-loop ensemble PSF fitting accurately determined microtubule diameter from STED images.
  • Successfully measured the diameter of endoplasmic reticulum tubules in live COS-7 cells.

Conclusions:

  • This technique provides a reliable way to measure resolution in situ for various microscopy techniques.
  • The algorithm, available as a plugin for the Python Microscopy Environment, enables accurate cellular feature size extraction.