Mark A. Haidekker

Molecular rotors—fluorescent biosensors for viscosity and flow

Viscosity is a measure of the resistance of a fluid against gradients in flow (shear rate). Both flow and viscosity play an important role in all biological systems from the microscopic (e.g., cellular) to the systemic level. Many methods to measure viscosity and flow have drawbacks, such as the tedious and time-consuming measurement process, expensive instrumentation, or the restriction to bulk sample sizes. Fluorescent environment-sensitive dyes are known to show high sensitivity and high spatial and temporal resolution. Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation and therefore exhibit two competing deexcitation pathways: fluorescence emission and non-radiative deexcitation from the TICT state. Since TICT formation is viscosity-dependent, the emission intensity of molecular rotors depends on the solvents viscosity. Furthermore, shear-stress dependency of the emission intensity was recently described. Although the photophysical processes are widely explored, the practical application of molecular rotors as sensors for viscosity and the fluid flow introduce additional challenges. Intensity-based measurements are influenced by fluid optical properties and dye concentration, and solvent-dye interaction requires calibration of the measurement system to a specific solvent. Ratiometric dyes and measurement systems help solve these challenges. In addition, the combination of molecular rotors with specific recognition groups allows them to target specific sites, for example the cell membrane or cytoplasm. Molecular rotors are therefore emerging as new biosensors for both bulk and local microviscosity, and for flow and fluid shear stress on a microscopic scale and with real-time response.

Environment-sensitive behavior of fluorescent molecular rotors

Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation. When intramolecular twisting occurs, the molecular rotor returns to the ground state either by emission of a red-shifted emission band or by nonradiative relaxation. The emission properties are strongly solvent-dependent, and the solvent viscosity is the primary determinant of the fluorescent quantum yield from the planar (non-twisted) conformation. This viscosity-sensitive behavior gives rise to applications in, for example, fluid mechanics, polymer chemistry, cell physiology, and the food sciences. However, the relationship between bulk viscosity and the molecular-scale interaction of a molecular rotor with its environment are not fully understood. This review presents the pertinent theories of the rotor-solvent interaction on the molecular level and how this interaction leads to the viscosity-sensitive behavior. Furthermore, current applications of molecular rotors as microviscosity sensors are reviewed, and engineering aspects are presented on how measurement accuracy and precision can be improved.

Temporal Gradients in Shear, but Not Spatial Gradients, Stimulate Endothelial Cell Proliferation

Background—The effect of temporal and spatial gradients in shear on primary human endothelial cell (HUVEC) proliferation was investigated. The sudden-expansion flow chamber (SEFC) model was used to differentiate the effect of temporal gradients in shear from that of spatial gradients. With a sudden onset of flow, cells are exposed to both temporal and spatial gradients of shear. The temporal gradients can be eliminated by slowly ramping up the flow. Methods and Results—HUVEC proliferation in the SEFC remained unstimulated when the onset of flow was slowly ramped. Sudden onset of flow stimulated a 105% increase of HUVEC proliferation (relative to ramped onset) within the region of flow reattachment. To further separate temporal and spatial gradients, a conventional parallel-plate flow chamber was used. A single 0.5-second impulse of 10 dyne/cm2 increased HUVEC proliferation 54±3% relative to control. When flow was slowly ramped over 30 seconds, HUVEC proliferation was not significantly different from controls. Steady laminar shear over 20 minutes inhibited HUVEC proliferation relative to controls regardless of step (36±8%) or ramp (21±5%) onsets of flow. Conclusions—The results indicate that temporal gradients in shear stress stimulate endothelial cell proliferation, whereas spatial gradients affect endothelial proliferation no differently than steady uniform shear stress.

New fluorescent probes for the measurement of cell membrane viscosity

BACKGROUNDnMolecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility.nnnRESULTSnLonger hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses.nnnCONCLUSIONSnThe modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

Dyes with Segmental Mobility: Molecular Rotors

Molecular rotors are fluorescent molecules that are characterized by the ability to form twisted states through the rotation of one segment of the structure with respect to the rest of the molecule. Intramolecular rotation changes the ground- state and excited-state energies, and molecular rotors deexcite from the twisted state either without photon emission or with a different wavelength than from the LE state. Intramolecular rotation is strongly dependent on the solvent. Solvent polarity, hydrogen bond formation, isomerization, excimer formation, and steric hindrance are predominant forms of solvent-fluorophore interaction. Of highest importance is sterichindrance,becauseitlinksthesolventsmicroviscositytotheformationrateof TICT states, which, in turn, determines the spectral emission. For this reason, molecular rotors have found a wide range of applications as fluorescent sensors of microviscosity and solvent free volume. Application examples include bulk viscos- ity measurement, probing dynamics of polymer formation, protein sensing and probing of protein aggregation, and microviscosity probing in living cells.

Relationship Between Structural Parameters, Bone Mineral Density and Fracture Load in Lumbar Vertebrae, Based on High-Resolution Computed Tomography, Quantitative Computed Tomography and Compression Tests

Abstract: Different noninvasive techniques for the assessment of the individual fracture risk in osteoporosis are introduced, and the relation between structural properties of high-resolution computed tomography (HR-CT) images of vertebral bodies, their bone mineral density (BMD) and the fracture load is analyzed. In 24 unfractured lumbar vertebrae with different degrees of demineralization from six specimens, the trabecular and cortical BMD was determined using quantitative CT. A lateral X-ray image revealed the number of fractures in the entire spine. A structural analysis of spongy and cortical bone was performed based on the HR-CT images. In the spongiosa, the fractal dimension was calculated as a function of the threshold value. In the cortical shell, the maximum number of clusters of low BMD was determined at varying threshold values. After the CT measurements the vertebrae were excised and compressed until fractured. On the basis of the spongiosa BMD and the number of fractures, 3 cases were found to be severely osteoporotic; the other 3 cases showed osteopenia. The average fracture loads were determined as 3533 N for the non-osteoporotic cases (range 2602–5802 N) and 1725 N for the osteoporotic cases (range 1311–2490 N). The parameters were determined as follows: average spongiosa BMD 115.2 mg/ml (101.8–135.3 mg/ml) for the non-osteoporotic cases, 46.2 mg/ml (34.8–57.6 mg/ml) for the osteoporotic cases; average cortical BMD 285.1 mg/ml (216.4–361.9 mg/ml) for the non-osteoporotic cases, 136.1 mg/ml (142.5–215.2 mg/ml) for the osteoporotic cases; spongiosa structure: average 0.5 (range 0.32–0.75) for the non-osteoporotic cases, average 1.05 (range 0.87–1.24) for the osteoporotic cases; cortical structure: average 81 (range 55–104) for the non-osteoporotic cases), average 136 (range 102–159) for the osteoporotic cases. Single parameters (BMD and structure) and weighted sums of these parameters were correlated with the fracture load, resulting in correlation coefficients of rsBMD= 0.82 (spongiosa BMD), rcBMD= 0.82 (cortical BMD), rsStr=–0.75 (spongiosa structure) and rcStr=–0.86 (cortical structure). The weighted sum of cortical and spongiosa BMD resulted in rBMD= 0.86, of cortical and spongiosa structure in rStr=–0.86. A weighted combination of all four parameters correlates with the fracture load at r4= 0.89, all correlations being statistically significant (p<0.0001). The four individual parameters show only a slight overlap between non-osteoporotic and osteoporotic subjects. The high correlation of the cortical BMD and the structural parameter in cortical bone indicates the important contribution of the cortical shell to vertebral stability. A weighted sum of multiple parameters results in a higher correlation with the fracture load and does not show an overlap between the two groups. It is best suited to estimate the individual fracture risk. The presented methods are generally applicable in vivo; and allow an improvement of the diagnosis of osteoporosis compared with the measurement of the BMD alone.

Rational Design of Amyloid Binding Agents Based on the Molecular Rotor Motif

Alzheimer’s disease (AD) is characterized by a progressive loss of cognitive function and constitutes the most common and fatal neurodegenerative disorder.[1] Genetic and clinical evidence supports the hypothesis that accumulation of amyloid deposits in the brain plays an important role in the pathology of the disease. This event is associated with perturbations of biological functions in the surrounding tissue leading to neuronal cell death, thus contributing to the disease process. The deposits are comprised primarily of amyloid (Aβ) peptides, a 39–43 amino acid sequence that self aggregates into a fibrillar β-pleated sheet motif. While the exact three-dimensional structure of the aggregated Aβ peptides is not known, a model structure that sustains the property of aggregation has been proposed.[2] This creates opportunities for in vivo imaging of amyloid deposits that can not only help evaluate the time course and evolution of the disease, but can also allow the timely monitoring of therapeutic treatments.[3]

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.

Synthesis and use of an in-solution ratiometric fluorescent viscosity sensor

A procedure for the synthesis of a ratiometric viscosity fluorescent sensor is described in this protocol. The essential requirement for the design of this sensor is the attachment of a primary fluorophore that has both a viscosity-independent fluorescence emission (coumarin dye shown in blue) and an emission from a fluorophore that exhibits viscosity-dependent fluorescent quantum yield (p-amino cinnamonitrile dye shown in red). The use of sensor 1 in viscosity measurements involves solubilization in a liquid of interest and excitation of the primary fluorophore at λex = 360 nm. The secondary fluorophore is simultaneously excited via resonance energy transfer. The ratio of the fluorescent emission of the secondary over the primary fluorophore provides a fast and precise measurement of the viscosity of the solvent. The synthesis of compound 1 using commercially available materials can be completed within 5 d.

NAD(P)H oxidase-mediated reactive oxygen species production alters astrocyte membrane molecular order via phospholipase A2.

ROS (reactive oxygen species) overproduction is an important underlying factor for the activation of astrocytes in various neuropathological conditions. In the present study, we examined ROS production in astrocytes and downstream effects leading to changes in the signalling cascade, morphology and membrane dynamics using menadione, a redox-active compound capable of inducing intracellular ROS. NAD(P)H oxidase-mediated menadione-induced ROS production, which then stimulated phosphorylation of p38 MAPK (mitogen-activated protein kinase) and ERK1/2 (extracellular-signal-regulated kinase 1/2), and increased actin polymerization and cytoskeletal protrusions. We also showed that astrocyte plasma membranes became more molecularly ordered under oxidative stress, which was abrogated by down-regulating cPLA2 (cytosolic phospholipase A2) either with a pharmacological inhibitor or by RNA interference. In addition, mild disruption of F-actin with cytochalasin D suppressed menadione-enhanced phosphorylation of cPLA2 and membrane alterations. Taken together, these results suggest an important role for ROS derived from NAD(P)H oxidase in activation of astrocytes to elicit biochemical, morphological and biophysical changes reminiscent of reactive astrocytes in pathological conditions.