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Related Concept Videos

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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Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes
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Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection

E M Boehm1, S Subramanyam1, M Ghoneim2

  • 1University of Iowa Carver College of Medicine, Iowa City, IA, United States.

Methods in Enzymology
|October 30, 2016
PubMed
Summary

Single-molecule total internal reflection fluorescence (TIRF) microscopy visualizes macromolecular complex formation. This technique helps understand cellular processes by analyzing how molecules associate and change structure.

Keywords:
Colocalization single-molecule imagingCompetitive and noncompetitive inhibitionKinetic event resolving algorithmProtein–protein and protein–nucleic acid interactionSingle-molecule fluorescenceTotal internal reflection fluorescence microscopy

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Area of Science:

  • Biophysics
  • Biochemistry
  • Cellular Biology

Background:

  • Macromolecular complexes are crucial for cellular functions.
  • Understanding their assembly and dynamics is key to deciphering cellular mechanisms.

Purpose of the Study:

  • To provide a comprehensive overview of single-molecule total internal reflection fluorescence (TIRF) microscopy.
  • To guide researchers in applying TIRF microscopy for studying macromolecular complex formation.

Main Methods:

  • Detailed discussion of single-molecule TIRF microscope setup and operation.
  • Strategies for immobilizing and fluorescently labeling macromolecules.
  • Application of one-color and multicolor TIRF imaging for binary and ternary complex analysis.

Main Results:

  • Demonstration of TIRF microscopy's capability to study macromolecular complex assembly.
  • Insights into the formation of binary and higher-order complexes.
  • Emphasis on experimental design, controls, data acquisition, and analysis.

Conclusions:

  • Single-molecule TIRF microscopy is a powerful tool for investigating macromolecular dynamics.
  • The study aims to broaden the accessibility and adoption of TIRF microscopy in biophysics and biochemistry.
  • Encourages the integration of TIRF microscopy into standard research methodologies.