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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.

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Related Experiment Video

Updated: May 16, 2026

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System
12:30

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Published on: February 9, 2017

Prism-type total internal reflection microscopy for single-molecule FRET.

Chirlmin Joo, Taekjip Ha

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

    Single-molecule fluorescence resonance energy transfer (FRET) allows detailed study of biological events. This protocol details prism-type total internal reflection microscopy for sensitive single-molecule FRET measurements.

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

    Published on: May 3, 2022

    Area of Science:

    • Biophysics
    • Molecular Biology
    • Microscopy

    Background:

    • Single-molecule (sm) fluorescence detection offers insights into biological processes without averaging.
    • Förster (fluorescence) resonance energy transfer (FRET) measures distances (30-80 Å) between molecules.
    • FRET changes can detect structural dynamics and molecular interactions.

    Purpose of the Study:

    • To describe the setup for prism-type total internal reflection (TIR) microscopy for single-molecule FRET (smFRET).
    • To provide a protocol for excitation and emission in prism-type TIR microscopy.

    Main Methods:

    • Utilizes prism-type total internal reflection (TIR) microscopy.
    • Focuses on the setup for excitation and emission pathways.
    • Applies to single-molecule fluorescence resonance energy transfer (smFRET) experiments.

    Main Results:

    • Establishes prism-type TIR microscopy as a key tool for smFRET.
    • Enables precise distance measurements in the 30-80 Å range.
    • Facilitates detection of molecular structural changes and interactions.

    Conclusions:

    • Prism-type TIR microscopy provides a robust platform for advanced smFRET studies.
    • This protocol aids researchers in implementing sensitive molecular detection techniques.
    • The method is crucial for understanding molecular mechanisms at the single-molecule level.