Fluorescence Lifetime Imaging for viscosity and diffusion measurements

摘要

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