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Cell surface fluctuations studied with defocusing microscopy.

U Agero1, C H Monken, C Ropert

  • 1Departamento de Física, ICEX, Universidade Federal de Minas Gerais, Caixa Postal 702, Belo Horizonte, CEP 30123-970 Minas Gerais, Brazil.

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
|June 6, 2003
PubMed
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Defocusing microscopy reveals cell surface curvature, offering insights into macrophage mechanics and phagocytosis. This technique quanties cytoskeleton dynamics, impacting cellular processes.

Area of Science:

  • Biophysics
  • Cell Biology
  • Optical Microscopy

Background:

  • Phase objects are typically invisible under standard microscopy.
  • Defocusing microscopy is an underutilized technique for visualizing phase objects.
  • Standard phase-contrast microscopy provides information on object thickness.

Purpose of the Study:

  • To revisit and apply defocusing microscopy theory to infinity-corrected optical microscopes.
  • To establish defocusing microscopy as a tool for imaging object curvature.
  • To investigate macrophage surface dynamics and their relation to phagocytosis.

Main Methods:

  • Developed a theoretical model where image contrast is proportional to the 2D Laplacian of phase difference.
  • Utilized artificial phase objects to validate the defocusing microscopy model.

Related Experiment Videos

  • Applied defocusing microscopy to study macrophage surface ruffles and cytoskeleton dynamics.
  • Employed optical tweezers to manipulate zymosan particles for phagocytosis studies.
  • Administered cytochalasin D to observe its effects on cytoskeleton and phagocytosis.

    • Main Results:

    • Defocusing microscopy images the curvature (2D Laplacian of thickness) of uniform phase objects.
    • Measured contrasts from artificial objects closely matched theoretical predictions.
    • Observed and quantified large, coherent, propagating structures (ruffles) on macrophage surfaces.
    • Correlated cytoskeleton fluctuations (decay time, correlation length) with viscoelastic properties.
    • Cytochalasin D inhibited ruffles, increased relaxation times, and prolonged phagocytosis.
    • Demonstrated a link between cytoskeleton motility and phagocytosis efficiency.

    Conclusions:

    • Defocusing microscopy provides a valuable method for visualizing and quantifying object curvature.
    • Macrophage surface ruffles are linked to cytoskeleton dynamics and mechanical properties.
    • Cytoskeleton motility plays a crucial role in the phagocytosis process.
    • The developed defocusing microscopy methods can assess the importance of cytoskeleton dynamics in cellular functions.