Live-cell fluorescence microscopy with molecular biosensors: what are we really measuring?

Biophys J. 2012 May 2;102(9):2003-11. doi: 10.1016/j.bpj.2012.03.055.


Engineered protein biosensors, such as those based on Förster resonance energy transfer, membrane translocation, or solvatochromic shift, are being used in combination with live-cell fluorescence microscopy to reveal kinetics and spatial localization of intracellular processes as they occur. Progress in the application of this approach has been steady, yet its general suitability for quantitative measurements remains unclear. To address the pitfalls of the biosensor approach in quantitative terms, simple reaction-diffusion models were analyzed. The analysis shows that although high-affinity molecular recognition allows robust detection of the fluorescence readout, either of two detrimental effects is fostered. Binding of an intramolecular biosensor or of a relatively abundant intermolecular biosensor introduces observer effects in which the dynamics of the target molecule under study are significantly perturbed, whereas binding of a sparingly expressed intermolecular biosensor is subject to a saturation effect, where the pool of unbound biosensor is significantly depleted. The analysis explores how these effects are manifest in the kinetics and spatial gradients of the biosensor-target complex. A sobering insight emerges: the observer or saturation effect is always significant; the question is whether or not it can be tolerated or accounted for. The challenge in managing the adverse effects is that specification of the biosensor-target affinity to within a certain order of magnitude is required.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Humans
  • Microscopy, Fluorescence / methods*
  • Molecular Imaging / methods*
  • Molecular Probe Techniques*
  • Molecular Probes / metabolism*
  • Protein Interaction Mapping / methods*


  • Molecular Probes