Variations in fluid viscosity are linked to a variety of functions and diseases both at the cellular level (e.g., membrane and cytoplasmic viscosity changes in cell signaling modulation) 1 and at the organismal level (e.g., blood, plasma, or lymphatic fluid viscosity changes in diabetes, hypertension, infarction, and aging). 2 It has been proposed that monitoring of biofluid viscosity could provide a diagnostic tool for the detection of diseases. 3 Since mechanical devices do not provide the spatial and temporal resolution needed, a new type of fluorescent-based viscosity sensors was developed. 4 These sensors are based on a class of environmentsensitive fluorescent dyes that are characterized by a viscositydependent emission quantum yield. 4,5 The chemical structure of these dyes contains an electron donor unit (such as a nitrogen atom) in conjugation with an electron acceptor unit (such as a nitrile). Upon photoexcitation, the two units can rotate relative to each other in a manner that is dependent on the viscosity of their environment. Representative examples of such fluorescent rotors are 9-(dicyanoVinyl)julolidine (DCVJ, 1) and 2-cyano-3-(4-dimethylaminophenyl)acrylic acid methyl ester (CMAM, 2) ( Figure 1). 5 Their viscosity-dependent fluorescent quantum yield is described by the Förster-Hoffmann equation (eq 1). 6 Fluorescent molecular rotors have been used for viscosity studies that are performed by steady-state fluorescence through emission intensity measurements. This method suffers, however, from drawbacks arising from changes of the fluid optical properties and fluctuations in dye concentrations. An additional disadvantage is that a calibration curve is needed for the absolute determination of viscosity. 5 As a consequence, changes in fluid properties and dye concentration may cause erroneous readings.We hypothesized that a dual dye composed of two distinct fluorescent units, one providing an internal intensity reference and the other acting as a viscosity sensor, would create a ratiometric sensing system, thus overcoming the above disadvantages. Dividing the sensor emission intensity by the reference emission intensity would yield a normalized intensity that should not only eliminate some of the fluid-and concentration-related artifacts but also provide a means to quantify viscosity by an internal reference. To test this hypothesis, we synthesized a compound 4 in which the CMAM motif was coupled with 7-methoxycoumarin-3-carboxylic acid (MCCA, 3). We chose MCCA as the donor fluorophore in order to induce excitation of the rotor moiety (CMAM) via Resonance Energy Transfer (RET). 7 We envisioned that, due to its viscosity-independent quantum yield, MCCA could be used as both the internal reference and the RET donor. The latter event could then excite the CMAM motif, resulting in a viscosity-dependent emission of the rotor. The linker was chosen to maintain a distance between the chromophores in the same range as the Förster distance 7a to allow considerable energy transfer to the acceptor combined w...