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Total Internal Reflection Fluorescence Microscopy01:05

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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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Related Experiment Video

Updated: Apr 5, 2026

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy
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Spinning-Spot Shadowless TIRF Microscopy.

Kyle L Ellefsen1, Joseph L Dynes2, Ian Parker3

  • 1Department of Neurobiology & Behavior, University of California Irvine, Irvine, CA, 92697, United States of America.

Plos One
|August 27, 2015
PubMed
Summary

This study introduces a novel Total Internal Reflection Fluorescence (TIRF) microscopy technique using spinning laser spots for uniform illumination. This method enhances imaging of near-membrane cellular processes and calcium dynamics.

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Total Internal Reflection Fluorescence (TIRF) microscopy visualizes near-membrane cellular events.
  • Standard TIRF implementations suffer from uneven illumination due to interference and scattering.
  • This limits the resolution and clarity of dynamic cellular processes.

Purpose of the Study:

  • To develop a method for achieving uniform illumination in TIRF microscopy.
  • To overcome limitations of conventional TIRF imaging.
  • To enable high-resolution, dynamic imaging of cellular functions.

Main Methods:

  • Utilized galvanometer-driven mirrors to rapidly spin the excitation laser spot in a circle.
  • Implemented computer control for precise scanning at 200 Hz with 5ms camera exposure.
  • Enabled adjustable scan radii for simultaneous TIRF, widefield, and skimming plane imaging modes.

Main Results:

  • Achieved effectively uniform illumination fields by averaging out irregularities.
  • Demonstrated dynamic recording of inositol trisphosphate-mediated Ca2+ signals.
  • Visualized STIM and Orai protein redistribution during store-operated Ca2+ entry.

Conclusions:

  • The spinning laser spot system significantly improves TIRF imaging quality.
  • This technique is versatile for various near-membrane studies, especially those involving fast dynamics.
  • The method offers enhanced capabilities for studying calcium signaling and protein dynamics.