Imaging viscosity and its spatiotemporal patterns can provide valuable insight into the underlying physical conditions of biochemical reactions and biological processes in cells and tissues. One way to measure viscosity and diffusion is the use of fluorescence recovery after photobleaching (FRAP). We combine FRAP with FLIM and time-resolved fluorescence anisotropy imaging (tr-FAIM), by acquiring time- and polarization-resolved fluorescence images in every frame of a FRAP series. This allows us to simultaneously monitor translational and rotational diffusion. This approach can be applied to measuring diffusion in homogeneous and heterogeneous environments, and in principle also allows the study of homo-FRET. Another way to measure viscosity and diffusion is through specific flexible dyes, e.g. fluorescent molecular rotors, whose fluorescence quantum yield and fluorescence lifetime depend on the viscosity of the environment, in combination with fluorescence lifetime imaging (FLIM). We show that a bodipybased fluorescent molecular rotor targeting mitochondria reports on their viscosity, which changes under physiological stimuli. Both methods can optically measure viscosity and diffusion on the micrometer scale.

Original languageEnglish
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XIX
EditorsAmmasi Periasamy, Peter T. C. So, Karsten Konig
ISBN (Electronic)9781510624061
Publication statusPublished - 22 Feb 2019
EventMultiphoton Microscopy in the Biomedical Sciences XIX 2019 - San Francisco, United States
Duration: 3 Feb 20196 Feb 2019


ConferenceMultiphoton Microscopy in the Biomedical Sciences XIX 2019
Country/TerritoryUnited States
CitySan Francisco


  • Fluorescence lifetime imaging
  • Fluorescence recovery after photobleaching
  • Fluorescent molecular rotors
  • Time-correlated single photon counting
  • Time-resolved fluorescence anisotropy imaging


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