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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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: May 23, 2026

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis
10:43

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Published on: November 6, 2019

Generating live cell data using total internal reflection fluorescence microscopy.

Derek Toomre

    Cold Spring Harbor Protocols
    |April 5, 2012
    PubMed
    Summary
    This summary is machine-generated.

    Total internal reflection fluorescence microscopy (TIRFM) offers exquisite sensitivity for imaging dynamic cellular processes near the cell cortex. This technique enables detailed visualization of cytoskeleton dynamics and membrane trafficking events at the single-molecule level.

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

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    Last Updated: May 23, 2026

    Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis
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    Published on: November 6, 2019

    Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy
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    Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy
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    Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

    Published on: January 21, 2019

    Area of Science:

    • Cell Biology
    • Microscopy Techniques
    • Biophysics

    Background:

    • Live cell fluorescent microscopy is crucial for understanding dynamic cellular processes.
    • Confocal microscopy offers improved resolution over epifluorescence, but Total Internal Reflection Fluorescence Microscopy (TIRFM) provides superior sensitivity for near-membrane events.
    • TIRFM utilizes an evanescent field to selectively excite fluorophores within a thin optical plane (<100 nm).

    Purpose of the Study:

    • To discuss the principles and applications of TIRFM in live cell imaging.
    • To provide guidance on sample preparation, time-lapse movie acquisition, and quantitative analysis for TIRFM.
    • To highlight TIRFM's utility in studying cytoskeleton dynamics and membrane trafficking (exo- and endocytosis).

    Main Methods:

    • Total Internal Reflection Fluorescence Microscopy (TIRFM) with high numerical-aperture (NA) objectives.
    • Quantitative analysis of time-lapse movies.
    • Sample preparation techniques optimized for TIRFM.

    Main Results:

    • TIRFM provides exquisite sensitivity for imaging cellular processes occurring near the cell cortex.
    • The technique allows for single-molecule, vesicle, or organelle level quantitation of dynamic events.
    • Examples demonstrate successful imaging of cytoskeleton dynamics and exo-/endocytosis.

    Conclusions:

    • TIRFM is an increasingly accessible and powerful tool for live cell imaging, particularly for processes at the cell periphery.
    • Quantitative TIRFM, combined with genetic and molecular methods, offers deep insights into cellular dynamics.
    • The accessibility of turnkey TIRFM systems facilitates its adoption by a broader range of biologists.