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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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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Attenuated total reflectance (ATR) infrared spectroscopy is a powerful analytical technique used to study the composition of materials. It is widely employed in chemistry, materials science, forensic science, and other fields where sample characterization is required. ATR has several advantages over traditional transmission IR spectroscopy, including the requirement of little to no sample preparation and the ability to analyze a wide range of samples.
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Updated: Mar 21, 2026

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
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Single-Molecule Total Internal Reflection Fluorescence Microscopy.

Emily M Kudalkar1, Trisha N Davis1, Charles L Asbury2

  • 1Department of Biochemistry, University of Washington, Seattle, Washington 98195;

Cold Spring Harbor Protocols
|May 4, 2016
PubMed
Summary
This summary is machine-generated.

Total internal reflection fluorescence (TIRF) microscopy enables real-time, single-molecule visualization of biological processes. This technique enhances signal-to-noise ratio, improving clarity for studying molecular motors and DNA replication.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Total internal reflection fluorescence (TIRF) microscopy offers unprecedented resolution for biological imaging.
  • It enables visualization of dynamic events at the single-molecule level, crucial for understanding complex cellular mechanisms.

Purpose of the Study:

  • To introduce the fundamental principles and advantages of TIRF microscopy.
  • To highlight its utility in studying dynamic biological processes and molecular interactions.

Main Methods:

  • Utilizes the phenomenon of total internal reflection to excite fluorophores only in a thin layer near the coverslip.
  • Minimizes background fluorescence, significantly enhancing the signal-to-noise ratio for single-molecule detection.

Main Results:

  • TIRF microscopy allows real-time observation of molecular motors, DNA replication, and membrane dynamics like endocytosis and exocytosis.
  • Improved visualization of cytoskeletal structures and dynamics at the plasma membrane.

Conclusions:

  • TIRF microscopy is a powerful and increasingly accessible technique for single-molecule investigations.
  • Its ability to provide high-resolution, real-time data makes it invaluable for cell biology and biophysics research.