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Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Updated: Dec 3, 2025

Synthesis and Characterization of Supramolecular Colloids
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Developing Scattering Morphology Resolved Total Internal Reflection Microscopy (SMR-TIRM) for Orientation Detection

Aidin Rashidi1, Sergio Domínguez-Medina1, Jiarui Yan2

  • 1Chemical and Biomolecular Engineering Department, Case Western Reserve University, 2102 Adelbert Road, Cleveland, Ohio 44106, United States.

Langmuir : the ACS Journal of Surfaces and Colloids
|October 26, 2020
PubMed
Summary

This study introduces scattering morphology resolved total internal reflection microscopy (SMR-TIRM) to measure interactions of anisotropic colloidal particles. SMR-TIRM can resolve particle orientation and separation distance, crucial for understanding colloidal behavior.

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Area of Science:

  • Colloid and surface science
  • Soft matter physics
  • Optical microscopy techniques

Background:

  • Anisotropic colloidal particles are crucial in various applications.
  • Measuring interactions of anisotropic particles at the ~kT scale is challenging.
  • Existing techniques lack precise orientation and separation distance measurements for anisotropic colloids.

Purpose of the Study:

  • To develop and validate a novel technique, scattering morphology resolved total internal reflection microscopy (SMR-TIRM).
  • To demonstrate SMR-TIRM's capability in measuring anisotropic particle orientation and interactions.
  • To enable accurate calculation of potential energy landscapes for colloidal ellipsoids.

Main Methods:

  • Development of SMR-TIRM for analyzing light scattered from anisotropic particles.
  • Utilizing an evanescent wave to probe particle-fluid interactions.
  • Fitting scattered light morphologies with 2D Gaussian surfaces to extract orientation and aspect ratio parameters.
  • Comparing experimental and simulation data for colloidal ellipsoids at varied orientations.

Main Results:

  • Scattering morphology orientation (Mφ) correlates with particle azimuthal angle.
  • Scattering morphology aspect ratio (M_AR) is sensitive to particle polar angle.
  • Both azimuthal and polar angles are resolved effectively, outperforming bright-field microscopy.
  • Integrated scattering intensity is orientation-dependent, requiring careful interpretation.

Conclusions:

  • SMR-TIRM successfully resolves both azimuthal and polar angles of colloidal ellipsoids.
  • A method is established to interpret orientation-dependent scattering intensity for accurate separation distance determination.
  • This technique provides a pathway to precisely map the potential energy landscapes of anisotropic colloids.