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Related Concept Videos

Atomic Fluorescence Spectroscopy01:29

Atomic Fluorescence Spectroscopy

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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...
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Confocal Fluorescence Microscopy01:16

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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Total Internal Reflection Fluorescence Microscopy01:05

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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 Microscopy01:37

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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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Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.
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Related Experiment Video

Updated: Apr 28, 2026

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Scanning inverse fluorescence correlation spectroscopy.

Jan Bergstrand, Daniel Rönnlund, Jerker Widengren

    Optics Express
    |June 13, 2014
    PubMed
    Summary

    Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) accurately measures nanodomain sizes on surfaces. This technique provides precise measurements for nanodomains and protein clusters in cell membranes.

    Area of Science:

    • Biophysics
    • Surface Science
    • Microscopy

    Background:

    • Nanodomains and protein clusters are crucial in cellular functions.
    • Accurate sizing of these nanoscale structures is essential for understanding biological processes.
    • Current methods for nanodomain sizing have limitations.

    Purpose of the Study:

    • Introduce Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) for absolute nanodomain size determination.
    • Develop and validate equations for calculating domain size using siFCS.
    • Demonstrate the application of siFCS on model surfaces and discuss its potential for biological samples.

    Main Methods:

    • Development of equations for domain size calculation from cross- and auto-correlation functions.
    • Measurement simulations to validate the derived equations.

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  • Experimental measurements on model surfaces using a confocal microscope with 270 nm resolution.

    • Main Results:

    • siFCS accurately estimated the size of 250 nm domains to be 257 ± 12 nm in diameter.
    • The technique estimated 40 nm domains to be 65 ± 26 nm in diameter.
    • Simulations and experimental data confirmed the validity of the siFCS method for nanodomain sizing.

    Conclusions:

    • siFCS is a robust method for determining the absolute size of nanodomains on surfaces.
    • The technique shows promise for sizing nanodomains and protein clusters within cell membranes.
    • siFCS offers a valuable tool for nanoscale research in biophysics and cell biology.