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.
Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ϵ, is around 5 in lipid droplets and 25<ϵ<40 in other lipid-rich regions. Our spectrally resolved fluorescence lifetime imaging microscopy (FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.
Meso-substituted boron-dipyrromethene (BODIPY-C12) was used to monitor the viscosity in cells via fluorescence
lifetime imaging (FLIM), and time-resolved fluorescence anisotropy measurements. Our results show that meso-substituted
BODIPY-C12 senses the viscosity in HeLa cells and is insensitive to the surrounding polarity. The
relationship between the fluorescence lifetime and the rotational correlation time of the dye in homogeneous solutions
agree with the combination of the Foerster Hoffmann equation and the Debye-Stokes-Einstein equation.
We present fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging along with translational diffusion
measurements of living cells labelled with green fluorescent protein (GFP) recorded in a single experiment. The
experimental set-up allows for time and polarization-resolved fluorescence images to be measured in every frame of a
fluorescence recovery after photobleaching (FRAP) series. We have validated the method using rhodamine 123 in
homogeneous solution prior to measurements of living A431 cells labelled with cdc42-GFP, for which the FRAP
recovery exhibits an immobile fraction and the rotational mobility of the protein is hindered while the fluorescence
lifetime fairly homogeneous across the cell. By eliminating the need for sequential measurements to extract fluorescence
lifetimes and molecular diffusion coefficients we remove artefacts arising from changes in sample morphology and
excessive photobleaching during sequential experiments.
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