A number of diseases and malfunctions at the cellular level can be attributed, at least in part, to changes in viscosity —one of the primary determinants of the diffusion rate in condensed media — which is integral to metabolic processes, signaling and transport. A number of studies have reported methods for measuring bulk macroscopic viscosity. However, because they typically require large sample volumes, these methods “are not suitable for mapping viscosity in heterogeneous samples,” said Marina K. Kuimova, a researcher with Imperial College London. “Working with biological samples, such as cells and tissues, is particularly challenging.”In a Journal of the American Chemical Society paper published May 28, 2008, Kuimova and collaborators at Imperial College London, at King’s College London and at PhotoBiotics Ltd., also in London, described a technique for imaging local microviscosity by taking advantage of the fluorescence lifetime of a molecular rotor. Molecular rotors, in which the nonradiative decay of the fluorescence excited state (and, hence, the fluorescence intensity) is affected by changes in viscosity, have proved promising as probes for local microviscosity. However, as fluorescence intensity also can be influenced by fluorophore concentration and the optical properties of the medium, calibration of intensity-based rotor responses can be difficult.Researchers have reported imaging local microviscosity by measuring the fluorescence lifetime of a “molecular rotor.” Changes in viscosity can contribute to a number of diseases and malfunctions at the cellular level. Shown here are images of cells incubated with a molecular rotor: a fluorescence intensity image (black and white), a typical fluorescence decay trace and a fluorescence lifetime image (color). The novelty of the present study, Kuimova said, is in using fluorescence lifetime instead of intensity and in combining lifetime detection with fluorescence lifetime imaging microscopy (FLIM). This approach determines lifetime with high precision and enables high-spatial-resolution viscosity maps of heterogeneous samples.The researchers designed a BODIPY-based fluorophore — the lifetime and intensity of which both change as a function of viscosity — and calibrated the viscosity-dependent responses. Then they incubated this rotor in living cells, using the human ovarian carcinoma cell line SK-OV-3, and acquired fluorescence-lifetime-based images with a Leica inverted scanning confocal microscope coupled with a Becker & Hickl time-correlated single-photon-counting card. A Hamamatsu pulsed diode laser operating at 467 nm, with a pulse duration of 90 ps and a repetition rate of 20 MHz, served as the source of excitation. Imaging was performed using a 63×, 1.2-NA water-immersion objective. A Becker & Hickl cooled detector, based on a Hamamatsu photomultiplier, collected the emission. The acquisition time was 200 s per image.The fluorescence lifetime images revealed a narrow distribution, between 1.4 and 1.8 ns, which, according to the researchers’ calibration graph, corresponded to an average viscosity of 140 ±40 cP. Thus they demonstrated direct measurement of intracellular viscosity with resolution comparable to that of a confocal microscope.Because the fluorescence lifetime of the molecular rotor also can be affected by binding to the intracellular targets, the researchers sought to ensure that the long fluorescence lifetime was caused by high environmental viscosity and not by binding or other types of quenching. They therefore used a complementary technique — time-resolved fluorescence anisotropy — to ensure that both measurements yielded similar values and, thus, to rule out other causes of the long fluorescence lifetime.Besides demonstrating the new technique, the investigators showed that the viscosity of cellular domains where the rotor localizes is significantly higher than that of water or cellular cytosol — roughly two orders of magnitude. This agrees with previous reports suggesting low average diffusion coefficient in a cell, which may be an important factor in processes involving diffusion of reactive species such as cytotoxic singlet oxygen; for example, increasing the efficiency of the processes by increasing the lifetimes.Competing techniquesKuimova noted that other techniques are available for measuring microviscosity. The first is the ratiometric approach, in which the probe consists of two parts — the molecular rotor and a fluorophore independent of viscosity that provides internal intensity reference. The synthesis and design of the rotor can be challenging, however, and its compatibility with biological samples should be considered.Time-resolved fluorescence anisotropy offers another means to estimate viscosity. “Any fluorophore with sufficiently long lifetime can be used for this,” Kuimova said. But she added that this approach is considerably more technically demanding than FLIM because it requires separate detection of fluorescence decays with vertical and horizontal polarization.The researchers are designing molecular rotor-based probes for exploring a variety of cellular domains. They plan to apply the technique, generally, to visualizing the intracellular environment and to gaining a better understanding of the processes that might affect cell function.