Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues. Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm. 6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects.In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.
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ProtocolThe protocols for FLIM sample preparation do not differ from those for confocal or wide-field intensity-based fluorescence microscopy. The data acquisition is followed by the main task of data analysis, i.e. extracting the fluorescence lifetimes from the raw data. Once these have been obtained, data interpretation helps to verify or falsify hypotheses.
Staining cells with molecular rotors1. Prepare a stock solution (10 ml) by dissolving approximately 1 mg/ml of the dye in an appropriate solvent (e.g. methanol for BODIPY-C 12 ) 6,7 using an accurate balance and a pipette. 2. The cells (a model cancer cell line, HeLa in our case) to be stained are grown on in a multiwell plate with a coverslide underside for microscopy, in an incubator at 37 °C with a 5 % CO 2 atmosphere until ~ 80% confluent. 3. Add 10 -20 μl of the stock solution to the living cells growing in a multi-well plate (SmartSlide 50 micro-incubation system, Wafergen) in 4 ml of Opti-MEM medium (GIBCO) per well for a 6-well plate. This yields a micro-molar dye concentration in the well. 4. Return the multi-well plate to an incubator at 37 °C with a 5 % CO 2 atmosphere for 10-45 mins for staining. 5. Remove the multiwell plate from the incubator and wash the cells 3-4 times with 4 ml optically clear cell culture medium (e.g. Opti-MEM) to remove excess dye.