Related Experiment Video
Updated: Mar 6, 2026
11:26
Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy
Published on: September 8, 2009
9.9K
Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution
Gary C H Mo1, Brian Ross1,2, Fabian Hertel1
1Department of Pharmacology, University of California San Diego, La Jolla, California, USA.
Nature Methods
|March 14, 2017
Summary
Researchers developed new fluorescent biosensors (FLINC) to visualize dynamic protein activities in living cells. These sensors reveal cellular activity architecture with super-resolution, uncovering microdomains of protein kinase A activity.
Area of Science:
- Cell Biology
- Biophysics
- Molecular Imaging
Background:
- Cellular processes rely on compartmentalized biochemical activities.
- Existing methods lack the resolution to visualize dynamic protein activities at the scale of cellular compartments.
- A generalizable method for high-resolution visualization of cellular biochemical activities is needed.
Purpose of the Study:
- To introduce a new class of fluorescent biosensors (FLINC) for visualizing dynamic protein activities in living cells.
- To achieve visualization resolution beyond the diffraction limit.
- To investigate the spatial organization of biochemical activities within cells.
Main Methods:
- Development of FLINC (fluorescent imaging with non-photobleaching contrast) biosensors.
- Utilizing binding-induced changes in protein fluorescence dynamics.
- Quantifying fluorescence fluctuations via stochastic optical fluctuation imaging (SOFI).
- Employing a protein kinase A (PKA) biosensor for experimental validation.
Main Results:
- FLINC biosensors enable visualization of biochemical activities at resolutions up to threefold better than the diffraction limit.
- Minute PKA activity microdomains on living cell plasma membranes were resolved.
- The role of clustered anchoring proteins in organizing these PKA activity microdomains was uncovered.
- Demonstrated the ability to resolve the spatial organization of cellular biochemical activities.
Conclusions:
- FLINC biosensors provide a novel, generalizable method for high-resolution imaging of dynamic cellular activities.
- Cellular biochemical activities are spatially organized into an 'activity architecture'.
- These biosensors can reveal the structural and functional characteristics of this activity architecture.
Related Concept Videos
01:37Super-resolution Fluorescence Microscopy
14.7K
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...
14.7K
01:19Protein Dynamics in Living Cells
2.8K
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...
2.8K