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Related Concept Videos

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 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...
Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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,...
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.
Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

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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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-focus fluorescence correlation spectroscopy.

Christoph Pieper1, Kerstin Weiß, Ingo Gregor

  • 1Third Institute of Physics-Biophysics, Georg-August-University Göttingen, Göttingen, Germany.

Methods in Enzymology
|January 2, 2013
PubMed
Summary
This summary is machine-generated.

Dual-focus fluorescence correlation spectroscopy (2fFCS) accurately measures molecular diffusion and flow velocities. This advanced technique uses two focal spots to provide a precise length scale for quantitative analysis in solutions and membranes.

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Area of Science:

  • Biophysics
  • Physical Chemistry
  • Optical Spectroscopy

Background:

  • Fluorescence Correlation Spectroscopy (FCS) is a powerful tool for studying molecular dynamics.
  • Accurate determination of diffusion coefficients and flow velocities is crucial in various scientific fields.
  • Existing methods may face limitations in precision and accuracy for absolute measurements.

Purpose of the Study:

  • To introduce and explain the technique of dual-focus fluorescence correlation spectroscopy (2fFCS).
  • To highlight the advantages of 2fFCS for precise quantitative measurements.
  • To demonstrate its application in determining diffusion coefficients and flow velocities.

Main Methods:

  • Utilizes two laterally shifted, overlapping focal regions for fluorescence signal acquisition.
  • Employs auto- and cross-correlation analysis of fluorescence signals.
  • Leverages the precisely known distance between focal regions as an external length scale.

Main Results:

  • Enables accurate determination of diffusion coefficients for fluorescent molecules and particles.
  • Allows for noninvasive, three-dimensional measurement of flow-velocity profiles.
  • Provides absolute values of diffusivities and velocities at pico- to nanomolar concentrations.

Conclusions:

  • 2fFCS offers superior accuracy and precision for measuring diffusion and flow.
  • The technique's inherent length scale overcomes optical aberrations, ensuring reliable results.
  • 2fFCS is the preferred method for quantitative biophysical and chemical analyses.