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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.
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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 developed.
Atomic Force Microscopy01:08

Atomic Force Microscopy

Atomic force microscopy (AFM) is a type of scanning probe microscopy that can analyze topographic details of various specimens like ceramics, glass, polymers, and biological samples. AFM offers over 1000 times more resolution than the optical imaging system. Images generated from AFM are three-dimensional surface profiles, offering an advantage over the flat, two-dimensional images from other imaging techniques.
The AFM Probe
The probe is regarded as the heart of any AFM setup and comprises the...

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Related Experiment Video

Updated: May 16, 2026

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy
08:55

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

Published on: February 17, 2023

Single-molecule FRET with total internal reflection microscopy.

Chirlmin Joo, Taekjip Ha

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

    Single-molecule Förster resonance energy transfer (FRET) microscopy visualizes biological dynamics. This technique uses fluorescence energy transfer to measure molecular distances, offering insights into structural changes and interactions.

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    Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox
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    Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

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    Related Experiment Videos

    Last Updated: May 16, 2026

    Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy
    08:55

    Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

    Published on: February 17, 2023

    Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
    06:43

    Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

    Published on: May 3, 2022

    Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox
    07:12

    Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

    Published on: July 5, 2021

    Area of Science:

    • Biophysics
    • Molecular Biology
    • Microscopy

    Background:

    • Single-molecule (sm) fluorescence detection bypasses time and population averaging for detailed biological event analysis.
    • Förster (fluorescence) resonance energy transfer (FRET) quantifies distances (30-80 Å) between donor and acceptor molecules via energy transfer efficiency.

    Purpose of the Study:

    • To provide a comprehensive guide to single-molecule Förster resonance energy transfer (smFRET) microscopy.
    • To detail the practical aspects of setting up and performing smFRET experiments using total internal reflection (TIR) microscopy.

    Main Methods:

    • Focus on total internal reflection (TIR) microscopy for single-molecule fluorescence detection.
    • Discusses critical experimental steps including dye selection, nucleic acid and protein labeling, surface preparation, and data acquisition.
    • Covers various data analysis methods and practical considerations for setting up both objective-type and prism-type TIR microscopy.

    Main Results:

    • Demonstrates the utility of smFRET for detecting structural changes in biomolecules.
    • Highlights the capability of smFRET to reveal relative motion between interacting molecules.
    • Provides a framework for implementing smFRET experiments with TIR microscopy.

    Conclusions:

    • smFRET combined with TIR microscopy is a powerful approach for studying molecular dynamics at the single-molecule level.
    • The article serves as a practical resource for researchers aiming to utilize smFRET techniques.
    • Successful implementation requires careful consideration of labeling, surface chemistry, and microscopy setup.