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Updated: Aug 22, 2025

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions
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Single Protein Detection and Imaging with Evanescent Scattering Microscopy.

Pengfei Zhang1, Lei Zhou1,2, Rui Wang1

  • 1Biodesign Center for Bioelectronics and Biosensors, Arizona State University, Tempe, USA.

Bio-Protocol
|November 10, 2022
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Summary

This study presents a label-free method for imaging single proteins using scattered evanescent waves on standard cover glass. This technique enables quantification of protein-antibody interactions at the single-molecule level without specialized equipment.

Keywords:
Cover glassEvanescent fieldScattering microscopySingle proteinSingle-molecule imagingTotal internal reflection

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Biochemistry

Background:

  • Single-molecule measurements offer insights beyond ensemble averages.
  • Evanescent detection enhances light-analyte interaction for sensitive detection.
  • Existing methods often require specialized sensing components.

Purpose of the Study:

  • To develop a label-free single-molecule imaging method.
  • To enable protein-antibody interaction quantification.
  • To utilize conventional microscopy and consumables.

Main Methods:

  • Detecting evanescent waves scattered by single protein molecules.
  • Imaging proteins on a plain cover glass surface.
  • Utilizing label-free optical detection principles.

Main Results:

  • Successfully imaged individual proteins on a standard cover glass.
  • Quantified protein-antibody interactions at the single-molecule level.
  • Demonstrated a method compatible with commercial optical microscopes.

Conclusions:

  • Single-molecule imaging is achievable on plain surfaces using scattered evanescent waves.
  • This approach simplifies single-molecule detection protocols.
  • The method has potential for widespread adoption in biological research.