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Related Concept Videos

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,...
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.
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.

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Related Experiment Video

Updated: Jul 20, 2026

Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment
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Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope.

Daniel R Sisan1, Richard Arevalo, Catherine Graves

  • 1Department of Physics, Georgetown University, Washington, DC, USA.

Biophysical Journal
|September 5, 2006
PubMed
Summary

We developed a faster fluorescence correlation spectroscopy (FCS) method using spinning disk confocal microscopy. This technique spatially maps diffusion and flow at high resolution, enabling studies of complex biological environments.

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Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells
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Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Published on: November 12, 2020

Area of Science:

  • Biophysics
  • Microscopy techniques
  • Materials science

Background:

  • Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying molecular dynamics.
  • Existing FCS methods have limitations in speed and spatial resolution.
  • Advancements are needed to analyze complex biological systems like tumors and cell nuclei.

Purpose of the Study:

  • To extend fluorescence correlation spectroscopy (FCS) capabilities using spinning disk confocal microscopy.
  • To achieve high-speed, spatially resolved measurements of diffusion and flow.
  • To address limitations of current FCS techniques in complex media.

Main Methods:

  • Development of an extended fluorescence correlation spectroscopy (FCS) approach.
  • Utilizing a spinning disk confocal microscope with high-speed cameras (1000 Hz).
  • Implementing corrections for pixel size effects and photobleaching specific to spinning disk confocal microscopy.

Main Results:

  • Simultaneous spatial mapping of diffusion coefficients or flow velocities at up to 10^5 locations.
  • Enabling measurements of systems with diffusion coefficients as low as 10^-7 cm^2/s.
  • Demonstrated application to microspheres in Type I collagen, revealing complex diffusion patterns.

Conclusions:

  • Spinning disk confocal microscopy significantly enhances FCS speed and spatial resolution.
  • The developed method allows for detailed analysis of molecular diffusion in complex biological matrices.
  • This technique opens new avenues for investigating cellular and extracellular environments.