Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution

Nat Methods. 2017 Apr;14(4):427-434. doi: 10.1038/nmeth.4221. Epub 2017 Mar 13.


Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

MeSH terms

  • Biosensing Techniques / methods*
  • Cell Membrane / metabolism
  • Cyclic AMP-Dependent Protein Kinases / analysis
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Escherichia coli / genetics
  • Fluorescence Resonance Energy Transfer / methods
  • Fluorescent Dyes
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Microscopy / instrumentation
  • Microscopy / methods
  • Molecular Imaging / methods
  • Mutagenesis, Site-Directed
  • Protein Interaction Mapping / methods
  • Stochastic Processes


  • Fluorescent Dyes
  • Green Fluorescent Proteins
  • Cyclic AMP-Dependent Protein Kinases