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Phase Contrast and Differential Interference Contrast Microscopy01:26

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In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of...
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Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules
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Reflection interference contrast microscopy of arbitrary convex surfaces.

Jose C Contreras-Naranjo1, James A Silas, Victor M Ugaz

  • 1Artie McFerrin Department of Chemical Engineering, Jack E. Brown Engineering Building, Texas A&M University, College Station, Texas 77843-3122, USA.

Applied Optics
|July 22, 2010
PubMed
Summary

Reflection Interference Contrast Microscopy (RICM) now accurately analyzes unknown object geometries. This new method reconstructs convex surface shapes, advancing microscopy applications.

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Surface Science

Background:

  • Reflection Interference Contrast Microscopy (RICM) applications are typically limited to known geometries.
  • Accurate analysis of unknown object geometries using RICM often relies on approximated fringe spacing.

Purpose of the Study:

  • To develop an accurate RICM analysis method for unknown and nonplanar geometries.
  • To improve intensity calculation and modeling for arbitrary convex surfaces.

Main Methods:

  • Reviewed and improved intensity calculation formulation based on nonplanar interface image formation theory.
  • Developed a practical implementation method for wedges and convex surfaces.
  • Presented a RICM model for arbitrary convex surfaces, with or without uniform layers.

Main Results:

  • Experimental validation with polymer vesicles demonstrated accurate reconstruction of convex bottom shapes.
  • The improved RICM theory, calculation method, and surface model successfully fitted experimental intensity patterns.
  • Enabled accurate shape reconstruction for objects close to the substrate.

Conclusions:

  • The enhanced RICM approach overcomes limitations of known geometries.
  • Accurate reconstruction of convex object shapes is achievable with the new method.
  • This work expands the applicability of RICM in biophysics and surface science.