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Nanoscale cellular imaging with scanning angle interference microscopy.

Christopher DuFort1, Matthew Paszek2

  • 1Department of Surgery, University of California, San Francisco, California, USA; Department of Orthopaedic Surgery, University of California, San Francisco, California, USA.

Methods in Cell Biology
|June 30, 2014
PubMed
Summary

Scanning Angle Interference Microscopy (SAIM) offers nanometer precision for localizing fluorescent molecules along the optical axis. This technique is easily implemented on existing microscopes for advanced cell biology imaging.

Keywords:
CytoskeletonDynamicFocal adhesionInterferenceMembraneMicroscopyNanoscaleSuperresolution

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Optical Microscopy

Background:

  • Fluorescence microscopy is crucial for live-cell imaging with molecule-specific contrast.
  • High-resolution axial localization of fluorescent molecules remains a challenge.

Purpose of the Study:

  • To introduce Scanning Angle Interference Microscopy (SAIM) for high-resolution axial localization of fluorescent molecules.
  • To demonstrate SAIM's applicability for imaging cellular structures near a substrate.

Main Methods:

  • SAIM utilizes a reflective surface and a scanning excitation laser at varying incidence angles.
  • Interference patterns between incident and reflected light modulate emission intensity based on fluorophore height.
  • Measured intensities are fitted to an optical model for precise z-axis localization.

Main Results:

  • SAIM achieves 5-10 nm precision in localizing molecules along the optical axis.
  • The technique provides diffraction-limited lateral resolution.
  • SAIM successfully imaged cellular structures within 1 μm of the sample substrate.

Conclusions:

  • SAIM is a simple, high-resolution technique for axial localization of fluorescent molecules.
  • The method is easily adaptable to commercial total internal reflection fluorescence microscopes.
  • SAIM has significant potential for widespread application in cell biology research.