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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.
Atomic Fluorescence Spectroscopy01:29

Atomic Fluorescence Spectroscopy

Atomic fluorescence spectroscopy (AFS) is an analytical technique that involves the electronic transitions of atoms in a flame, furnace, or plasma being excited by electromagnetic (EM) radiation. When these atoms absorb energy, they become excited and subsequently release energy as they return to their original state. This emitted light, or "fluorescence," is observed at a right angle to the incident beam. Both absorption and emission processes transpire at distinct wavelengths, which are...
Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview01:13

Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview

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.
The ATR process begins by directing a beam...

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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Published on: December 11, 2021

Dual-color total internal reflection fluorescence cross-correlation spectroscopy.

Marcel Leutenegger, Hans Blom, Jerker Widengren

    Journal of Biomedical Optics
    |September 13, 2006
    PubMed
    Summary

    We developed a new dual-color total internal reflection fluorescence system for single-molecule analysis. This method enhances molecular brightness and is suitable for studying biochemical interactions at interfaces.

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    A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

    Published on: March 22, 2012

    Area of Science:

    • Biophysics
    • Analytical Chemistry
    • Molecular Biology

    Background:

    • Single-molecule analysis is crucial for understanding complex biochemical processes.
    • Fluorescence Cross-Correlation Spectroscopy (FCCS) enables the study of molecular interactions.
    • Limitations exist in conventional FCCS regarding sensitivity and efficiency.

    Purpose of the Study:

    • To develop and apply a novel dual-color total internal reflection (TIR) fluorescence system.
    • To enable advanced single-molecule coincidence analysis and FCCS.
    • To assess the system's performance in measuring biochemical binding assays.

    Main Methods:

    • Development of a dual-color TIR fluorescence system.
    • Application of the system to a synthetic DNA-binding assay.
    • Utilizing epi-illumination with a high numerical aperture for enhanced collection efficiency.
    • Employing global analysis for spectroscopic data interpretation.

    Main Results:

    • Demonstrated the suitability of the dual-color TIR-FCCS approach for coincidence assays.
    • Achieved a two- to three-fold increase in molecular brightness compared to confocal FCCS.
    • Successfully measured a synthetic DNA-binding assay at solid/liquid interfaces.
    • Validated the system for studying biochemical binding, fusion, and signal transduction.

    Conclusions:

    • The novel dual-color TIR-FCCS system offers enhanced sensitivity and efficiency for single-molecule studies.
    • This method is well-suited for investigating molecular interactions at interfaces.
    • The system provides a significant improvement over conventional FCCS techniques.