Molecular rotors are a class of fluorescent molecules that have been characterized in the 1970s to 1980s. Their main property is that they have two modes of relaxation. A molecule that reached its excited state through photon absorption can fall back into its ground state either by photon emission (fluorescence) or by intramolecular rotation, as can be seen in the picture below. Since the rotational relaxation is the preferred mode, molecular rotors are weakly fluorescent in its native state.
Intramolecular rotation can be inhibited if the molecule is dissolved in a fluid with low molecular free volume. Free volume is related to the fluid's viscosity (Doolittle, 1952). Therefore, the fluorescence quantum yield is related to the viscosity of the solvent. A mathematical relatioship exists, known as the Förster-Hoffmann-Equation (Förster & Hoffmann, 1971):
This equation (see a brief description of the derivation in Haidekker et al, Chem & Biochem 2001, p 127) shows that increased viscosity (eta) leads to increased quantum yield (phi) and thus to increased fluorescence intensity. x is a molecule-dependent constant (0.6 for DCVJ), and C is a temperature-dependent constant.
Using this physical property, the measurement of viscosity changes can be reduced to the simple measurement of fluorescence intensity changes.
|Emission spectra of DCVJ in three different media. It can be seen that glycerol, a very viscous fluid, causes a dramatically higher emission intensity than ethylene glycol, and that the intensity in a 50:50 mixture lies in between.|
State-of-the-art viscosity measurement of a viscous fluid involves shearing the fluid. The most widespread instruments are the falling- ball viscometer, the cone-and-plate viscometer, and the capillary viscometer. These mechanical methods have in common that a relatively large amount of fluid is needed, and that the measurement process takes time - generally up to several minutes. In addition, the instruments need to be meticulously cleaned.
The novel approach of using photophysical principles for viscosity measurement does not have these disadvantages. Fluorescence-based viscosity measurements can be taken with fluid volumes as low as some 100 microliters, and theoretically, fluorescence measurements take sub-second acquisition times. In addition, the use of molecular rotors allows the observation of microviscosity changes, limited merely by the optical resolution of the measurement instrument (microscope). For this reason, molecular rotors can be used in e.g. cell mechanotransduction research when observing changes in the fluidity of the cell membrane.
Research Project 1: The measurement of changes in cell membrane fluidity under fluid shear stress.
Blood vessels consist of several layers of cells. The innermost layer, the endothelium, is exposed to mechanical forces caused by the blood flow: Fluid shear stress. It has been shown that endothelial cells react to flow exposure in various ways, mainly in the activation of membrane-bound G-proteins and in the release of vasoactive substances, such as prostaglandin and nitric oxide. As a result, endothelial cells are an important part in the blood pressure control chain.
But how do endothelial cells sense fluid flow?
One hypothesis is that the membrane changes a physical property, the fluidity (fluidity = reciprocal of viscosity) and thus allows membrane-bound proteins to change their behavior. By using molecular rotors, membrane fluidity was monitored under different levels of fluid shear stress.
Emission intensity of endothelial cells stained with DCVJ under various levels of fluid shear stress. When the cell layer is exposed to fluid shear stress, there is a distinct drop in the emitted intensity. This indicates an increase in fluidity (=decrease in viscosity). Upon cessation of flow, the original level of intensity is restored, indicating that this form of membrane fluidization is reversible. Higher levels of shear stress lead to higher intensity drops. This suggests that membrane fluidization occurs in a dose-response manner.
The results of the experiments with cells exposed to shear stress strongly suggest that the cell membrane acts as an important cell mechanosensor and provides the first step in the signaling chain.
Research Project 2: Development of a fluorescence-based biofluid viscometer.
(detailed description will be posted soon)