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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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Microtubules01:35

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There are three types of cytoskeletal structures in eukaryotic cells—microfilaments, intermediate filaments, and microtubules. With a diameter of about 25 nm, microtubules are the thickest of these fibers. Microtubules carry out a variety of functions that include cell structure and support, transport of organelles, cell motility (movement), and the separation of chromosomes during cell division.
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The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary...
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Related Experiment Video

Updated: Jan 1, 2026

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy
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Studying Tau-Microtubule Interaction Using Single-Molecule TIRF Microscopy.

Virginie Stoppin-Mellet1, Nassiba Bagdadi2, Yasmina Saoudi2

  • 1Univ. Grenoble Alpes, Inserm, U1216, CNRS, Grenoble Institut Neurosciences, Grenoble, France. virginie.stoppin-mellet@univ-grenoble-alpes.fr.

Methods in Molecular Biology (Clifton, N.J.)
|December 28, 2019
PubMed
Summary

Researchers developed a new assay using Total Internal Reflection Fluorescence (TIRF) microscopy to study how Tau protein interacts with microtubules at the single-molecule level, revealing insights into microtubule dynamics.

Keywords:
Cell-free assaysMicrotubulesSingle moleculeTauTotal Internal Reflection Fluorescence microscopy

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Area of Science:

  • Cell Biology
  • Biophysics
  • Neuroscience

Background:

  • Microtubule (MT) architecture is regulated by microtubule-associated proteins (MAPs).
  • Understanding MAPs' interactions with MTs is crucial for deciphering cytoskeleton dynamics.
  • In vitro assays coupled with single-molecule fluorescence are powerful tools for this research.

Purpose of the Study:

  • To develop and validate a novel assay for characterizing Tau-microtubule interactions.
  • To enable single-molecule level analysis of Tau binding dynamics on dynamic microtubules.
  • To quantify Tau oligomerization states and their dwell times on microtubules.

Main Methods:

  • Utilized Total Internal Reflection Fluorescence (TIRF) microscopy.
  • Developed a protocol for protein sample preparation in flow cells.
  • Performed single-molecule acquisitions and quantitative analysis of Tau-microtubule interactions.

Main Results:

  • Successfully characterized Tau interaction with dynamic microtubules at the single-molecule level.
  • Quantified Tau oligomerization states and their dwell times on microtubules.
  • Demonstrated the utility of the TIRF-based assay for studying MAP-microtubule dynamics.

Conclusions:

  • The developed TIRF microscopy assay provides a robust platform for single-molecule analysis of Tau-microtubule interactions.
  • This method offers new perspectives for quantifying the dynamic behavior of MAPs on microtubules.
  • The findings contribute to a deeper understanding of microtubule regulation in biological systems.