Related Experiment Video
Updated: Aug 15, 2026
10:59
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
Published on: April 6, 2012
Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell
1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Cellular and Molecular Biology (Noisy-Le-Grand, France)
|October 9, 1998
Summary
Confocal fluorescence correlation spectroscopy (FCS) enables subcellular molecular interaction analysis. This study introduces fluorescence correlation microscopy (FCM) for precise intracellular measurements, detailing instrumental needs and data analysis.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Confocal fluorescence correlation spectroscopy (FCS) is valuable for analyzing molecular interactions within cells.
- Precise three-dimensional positioning is crucial for subcellular analysis using FCS.
- Existing techniques may lack the necessary precision for in-cell measurements.
Purpose of the Study:
- To develop and validate an instrument for intracellular FCS measurements.
- To establish instrumental requirements and data analysis methods for this technique.
- To introduce and define a new modality: fluorescence correlation microscopy (FCM).
Main Methods:
- Integration of FCS with high-sensitivity digital imaging microscopy.
- Implementation of high-precision three-dimensional positioning systems.
- Development of specific data analysis protocols for intracellular FCS.
Main Results:
- Successful execution of the first intracellular FCS measurements.
- Detailed specification of instrumental requirements for reliable measurements.
- Demonstration of the feasibility of fluorescence correlation microscopy (FCM).
Conclusions:
- The developed instrument enables precise intracellular molecular interaction analysis.
- Fluorescence correlation microscopy (FCM) is a viable extension of FCS for subcellular studies.
- This work lays the foundation for advanced in vivo molecular studies.
More Related Videos
Related Concept Videos
01:16Confocal 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,...
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
01:05Total 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.
01:37Super-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.