The importance of the epithelial fibre cell interface to lens regeneration in an in vivo rat model and in a human bag-in-the-lens (BiL) sample.

Human lens regeneration and the Bag-in-the-Lens (BIL) surgical treatment for cataract both depend upon lens capsule closure for their success. Our studies suggest that the first three days after surgery are critical to their long-term outcomes. Using a rat model of lens regeneration, we evidenced lens epithelial cell (LEC) proliferation increased some 50 fold in the first day before rapidly declining to rates observed in the germinative zone of the contra-lateral, un-operated lens. Cell multi-layering at the lens equator occurred on days 1 and 2, but then reorganised into two discrete layers by day 3. E- and N-cadherin expression preceded cell polarity being re-established during the first week. Aquaporin 0 (AQP0) was first detected in the elongated cells at the lens equator at day 7. Cells at the capsulotomy site, however, behaved very differently expressing the epithelial mesenchymal transition (EMT) markers fibronectin and alpha-smooth muscle actin (SMA) from day 3 onwards. The physical interaction between the apical surfaces of the anterior and posterior LECs from day 3 after surgery preceded cell elongation. In the human BIL sample fibre cell formation was confirmed by both histological and proteome analyses, but the cellular response is less ordered and variable culminating in Soemmerring's ring (SR) formation and sometimes Elschnig's pearls. This we evidence for lenses from a single patient. No bow region or recognisable epithelial-fibre cell interface (EFI) was evident and consequently the fibre cells were disorganised. We conclude that lens cells require spatial and cellular cues to initiate, sustain and produce an optically functional tissue in addition to capsule integrity and the EFI.

Neuronal SKN-1B modulates nutritional signalling pathways and mitochondrial networks to control satiety.

The feeling of hunger or satiety results from integration of the sensory nervous system with other physiological and metabolic cues. This regulates food intake, maintains homeostasis and prevents disease. In C. elegans, chemosensory neurons sense food and relay information to the rest of the animal via hormones to control food-related behaviour and physiology. Here we identify a new component of this system, SKN-1B which acts as a central food-responsive node, ultimately controlling satiety and metabolic homeostasis. SKN-1B, an ortholog of mammalian NF-E2 related transcription factors (Nrfs), has previously been implicated with metabolism, respiration and the increased lifespan incurred by dietary restriction. Here we show that SKN-1B acts in two hypothalamus-like ASI neurons to sense food, communicate nutritional status to the organism, and control satiety and exploratory behaviours. This is achieved by SKN-1B modulating endocrine signalling pathways (IIS and TGF-ß), and by promoting a robust mitochondrial network. Our data suggest a food-sensing and satiety role for mammalian Nrf proteins.

Three-dimensional data capture and analysis of intact eye lenses evidences emmetropia-associated changes in epithelial cell organization.

Organ and tissue development are highly coordinated processes; lens growth and functional integration into the eye (emmetropia) is a robust example. An epithelial monolayer covers the anterior hemisphere of the lens, and its organization is the key to lens formation and its optical properties throughout all life stages. To better understand how the epithelium supports lens function, we have developed a novel whole tissue imaging system using conventional confocal light microscopy and a specialized analysis software to produce three-dimensional maps for the epithelium of intact mouse lenses. The open source software package geometrically determines the anterior pole position, the equatorial diameter, and three-dimensional coordinates for each detected cell in the epithelium. The user-friendly cell maps, which retain global lens geometry, allow us to document age-dependent changes in the C57/BL6J mouse lens cell distribution characteristics. We evidence changes in epithelial cell density and distribution in C57/BL6J mice during the establishment of emmetropia between postnatal weeks 4-6. These epithelial changes accompany a previously unknown spheroid to lentoid shape transition of the lens as detected by our analyses. When combined with key findings from previous mouse genetic and cell biological studies, we suggest a cytoskeleton-based mechanism likely underpins these observations.

Endothelial TRPV4 channels modulate vascular tone by Ca2+ -induced Ca2+ release at inositol 1,4,5-trisphosphate receptors.

BACKGROUND AND PURPOSE: The TRPV4 ion channels are Ca2+ permeable, non-selective cation channels that mediate large, but highly localized, Ca2+ signals in the endothelium. The mechanisms that permit highly localized Ca2+ changes to evoke cell-wide activity are incompletely understood. Here, we tested the hypothesis that TRPV4-mediated Ca2+ influx activates Ca2+ release from internal Ca2+ stores to generate widespread effects. EXPERIMENTAL APPROACH: Ca2+ signals in large numbers (~100) of endothelial cells in intact arteries were imaged and analysed separately. KEY RESULTS: Responses to the TRPV4 channel agonist GSK1016790A were heterogeneous across the endothelium. In activated cells, Ca2+ responses comprised localized Ca2+ changes leading to slow, persistent, global increases in Ca2+ followed by large propagating Ca2+ waves that moved within and between cells. To examine the mechanisms underlying each component, we developed methods to separate slow persistent Ca2+ rise from the propagating Ca2+ waves in each cell. TRPV4-mediated Ca2+ entry was required for the slow persistent global rise and propagating Ca2+ signals. The propagating waves were inhibited by depleting internal Ca2+ stores, inhibiting PLC or blocking IP3 receptors. Ca2+ release from stores was tightly controlled by TRPV4-mediated Ca2+ influx and ceased when influx was terminated. Furthermore, Ca2+ release from internal stores was essential for TRPV4-mediated control of vascular tone. CONCLUSIONS AND IMPLICATIONS: Ca2+ influx via TRPV4 channels is amplified by Ca2+ -induced Ca2+ release acting at IP3 receptors to generate propagating Ca2+ waves and provide a large-scale endothelial communication system. TRPV4-mediated control of vascular tone requires Ca2+ release from the internal store.

VasoTracker, a Low-Cost and Open Source Pressure Myograph System for Vascular Physiology.

Pressure myography, one of the most commonly used techniques in vascular research, measures the diameter of isolated, pressurized arteries to assess the functional activity of smooth muscle and endothelial cells. Despite the widespread adoption of this technique for assessing vascular function, there are only a small number of commercial systems and these are expensive. Here, we introduce a complete, open source pressure myograph system and analysis software, VasoTracker, that can be set-up for approximately 10% of the cost of commercial alternatives. We report on the development of VasoTracker and demonstrate its ability to assess various components of vascular reactivity. A unique feature of the VasoTracker platform is the publicly accessible website (http://www.vasotracker.com/) that documents how to assemble and use this affordable, adaptable, and expandable pressure myograph.

Mitochondrial ATP production provides long-range control of endothelial inositol trisphosphate-evoked calcium signaling.

Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca2+ signaling or modify reactive oxygen species (ROS). To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.

Spatially structured cell populations process multiple sensory signals in parallel in intact vascular endothelium.

Blood flow, blood clotting, angiogenesis, vascular permeability, and vascular remodeling are each controlled by a large number of variable, noisy, and interacting chemical inputs to the vascular endothelium. The endothelium processes the entirety of the chemical composition to which the cardiovascular system is exposed, carrying out sophisticated computations that determine physiological output. Processing this enormous quantity of information is a major challenge facing the endothelium. We analyzed the responses of hundreds of endothelial cells to carbachol (CCh) and adenosine triphosphate (ATP) and found that the endothelium segregates the responses to these two distinct components of the chemical environment into separate streams of complementary information that are processed in parallel. Sensitivities to CCh and ATP mapped to different clusters of cells, and each agonist generated distinct signal patterns. The distinct signals were features of agonist activation rather than properties of the cells themselves. When there was more than one stimulus present, the cells communicated and combined inputs to generate new distinct signals that were nonlinear combinations of the inputs. Our results demonstrate that the endothelium is a structured, collaborative sensory network that simplifies the complex environment using separate cell clusters that are sensitive to distinct aspects of the overall biochemical environment and interactively compute signals from diverse but interrelated chemical inputs. These features enable the endothelium to selectively process separate signals and perform multiple computations in an environment that is noisy and variable.

Non-invasive in vivo quantification of the developing optical properties and graded index of the embryonic eye lens using SPIM.

Graded refractive index lenses are inherent to advanced visual systems in animals. By understanding their formation and local optical properties, significant potential for improved ocular healthcare may be realized. We report a novel technique measuring the developing optical power of the eye lens, in a living animal, by exploiting the orthogonal imaging modality of a selective plane illumination microscope (SPIM). We have quantified the maturation of the lenticular refractive index at three different visible wavelengths using a combined imaging and ray tracing approach. We demonstrate that the method can be used with transgenic and vital dye labeling as well as with both fixed and living animals. Using a key eye lens morphogen and its inhibitor, we have measured their effects both on lens size and on refractive index. Our technique provides insights into the mechanisms involved in the development of this natural graded index micro-lens and its associated optical properties.

Compact, modular and in-plane AOSLO for high-resolution retinal imaging.

The adaptive optics scanning laser ophthalmoscope (AOSLO) was first developed in 2002 and since then the technology has been adopted in several laboratories around the world, for both clinical and psychophysical research. There have been a few major design implementations of the AOSLO. The first used on-axis tilted spherical mirrors in a planar arrangement, and the second minimized the build up of astigmatism present in the first design by using a non-planar arrangement. Other designs have avoided astigmatism by using custom-made toroidal mirrors or by using lenses on-axis, rather than mirrors. We present a new design implementation for an AOSLO that maintains a planar optical alignment without the build up astigmatism using compact, reconfigurable modules based on an Offner relay system. We additionally use an off-the-shelf digital oscilloscope for data capture and custom-written Python code for generating and analyzing the retinal images. This design results in a compact system that is simple to align and, being composed of modular relays, has the potential for additional components to be added. We show that this system maintains diffraction-limited image quality across the field of view and that cones are clearly resolved in the central retina. The modular relay design is generally applicable to any system requiring one or more components in the pupil conjugate plane. This is likely to be useful for any point-scanned system, such as a standard scanning laser ophthalmoscope or non-ophthalmic confocal imaging system.

Dynamic lens and monovision 3D displays to improve viewer comfort.

Stereoscopic 3D (S3D) displays provide an additional sense of depth compared to non-stereoscopic displays by sending slightly different images to the two eyes. But conventional S3D displays do not reproduce all natural depth cues. In particular, focus cues are incorrect causing mismatches between accommodation and vergence: The eyes must accommodate to the display screen to create sharp retinal images even when binocular disparity drives the eyes to converge to other distances. This mismatch causes visual discomfort and reduces visual performance. We propose and assess two new techniques that are designed to reduce the vergence-accommodation conflict and thereby decrease discomfort and increase visual performance. These techniques are much simpler to implement than previous conflict-reducing techniques. The first proposed technique uses variable-focus lenses between the display and the viewer's eyes. The power of the lenses is yoked to the expected vergence distance thereby reducing the mismatch between vergence and accommodation. The second proposed technique uses a fixed lens in front of one eye and relies on the binocularly fused percept being determined by one eye and then the other, depending on simulated distance. We conducted performance tests and discomfort assessments with both techniques and compared the results to those of a conventional S3D display. The first proposed technique, but not the second, yielded clear improvements in performance and reductions in discomfort. This dynamic-lens technique therefore offers an easily implemented technique for reducing the vergence-accommodation conflict and thereby improving viewer experience.

Age decreases mitochondrial motility and increases mitochondrial size in vascular smooth muscle.

KEY POINTS: Age is proposed to be associated with altered structure and function of mitochondria; however, in fully-differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel-by-pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during 'flickers' of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly-elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly-motile. Mitochondria that are motile are located more peripherally in the cell than non-motile mitochondria. ABSTRACT: Mitochondrial function, motility and architecture are each central to cell function. Age-associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill-defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic 'flickers' of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker-assisted localization microscopy; FaLM). In freshly-isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X-Y areas in both young adult (3 months; 0.05-6.58 µm(2) ) and aged rats (18 months; 0.05-13.4 µm(2) ). In cells from young animals, most mitochondria were small (mode area 0.051 µm(2) ) compared to aged animals (0.710 µm(2) ). Cells from older animals contained a subpopulation of highly-elongated mitochondria (5.3% were >2 µm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 µm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 µm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian-like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 µm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly-elongated. These age-associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation.

Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Advancing Age Decreases Pressure-Sensitive Modulation of Calcium Signaling in the Endothelium of Intact and Pressurized Arteries.

Aging is the summation of many subtle changes which result in altered cardiovascular function. Impaired endothelial function underlies several of these changes and precipitates plaque development in larger arteries. The endothelium transduces chemical and mechanical signals into changes in the cytoplasmic calcium concentration to control vascular function. However, studying endothelial calcium signaling in larger arteries in a physiological configuration is challenging because of the requirement to focus through the artery wall. Here, pressure- and agonist-sensitive endothelial calcium signaling was studied in pressurized carotid arteries from young (3-month-old) and aged (18-month-old) rats by imaging from within the artery using gradient index fluorescence microendoscopy. Endothelial sensitivity to acetylcholine increased with age. The number of cells exhibiting oscillatory calcium signals and the frequency of oscillations were unchanged with age. However, the latency of calcium responses was significantly increased with age. Acetylcholine-evoked endothelial calcium signals were suppressed by increased intraluminal pressure. However, pressure-dependent inhibition of calcium signaling was substantially reduced with age. While each of these changes will increase endothelial calcium signaling with increasing age, decreases in endothelial pressure sensitivity may manifest as a loss of functionality and responsiveness in aging.

Flicker-assisted localization microscopy reveals altered mitochondrial architecture in hypertension.

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic "flickers" of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension.

Pressure-dependent regulation of Ca2+ signalling in the vascular endothelium.

KEY POINTS: Increased pressure suppresses endothelial control of vascular tone but it remains uncertain (1) how pressure is sensed by the endothelium and (2) how the vascular response is inhibited. This study used a novel imaging method to study large numbers of endothelial cells in arteries that were in a physiological configuration and held at normal blood pressures. Increased pressure suppressed endothelial IP3 -mediated Ca(2+) signals. Pressure modulated endothelial cell shape. The changes in cell shape may alter endothelial Ca(2+) signals by modulating the diffusive environment for Ca(2+) near IP3 receptors. Endothelial pressure-dependent mechanosensing may occur without a requirement for a conventional molecular mechanoreceptor. ABSTRACT: The endothelium is an interconnected network upon which haemodynamic mechanical forces act to control vascular tone and remodelling in disease. Ca(2+) signalling is central to the endothelium's mechanotransduction and networked activity. However, challenges in imaging Ca(2+) in large numbers of endothelial cells under conditions that preserve the intact physical configuration of pressurized arteries have limited progress in understanding how pressure-dependent mechanical forces alter networked Ca(2+) signalling. We developed a miniature wide-field, gradient-index (GRIN) optical probe designed to fit inside an intact pressurized artery that permitted Ca(2+) signals to be imaged with subcellular resolution in a large number (∼200) of naturally connected endothelial cells at various pressures. Chemical (acetylcholine) activation triggered spatiotemporally complex, propagating inositol trisphosphate (IP3 )-mediated Ca(2+) waves that originated in clusters of cells and progressed from there across the endothelium. Mechanical stimulation of the artery, by increased intraluminal pressure, flattened the endothelial cells and suppressed IP3 -mediated Ca(2+) signals in all activated cells. By computationally modelling Ca(2+) release, endothelial shape changes were shown to alter the geometry of the Ca(2+) diffusive environment near IP3 receptor microdomains to limit IP3 -mediated Ca(2+) signals as pressure increased. Changes in cell shape produce a geometric microdomain regulation of IP3 -mediated Ca(2+) signalling to explain macroscopic pressure-dependent, endothelial mechanosensing without the need for a conventional mechanoreceptor. The suppression of IP3 -mediated Ca(2+) signalling may explain the decrease in endothelial activity as pressure increases. GRIN imaging provides a convenient method that gives access to hundreds of endothelial cells in intact arteries in physiological configuration.

A dimensionless ordered pull-through model of the mammalian lens epithelium evidences scaling across species and explains the age-dependent changes in cell density in the human lens.

We present a mathematical (ordered pull-through; OPT) model of the cell-density profile for the mammalian lens epithelium together with new experimental data. The model is based upon dimensionless parameters, an important criterion for inter-species comparisons where lens sizes can vary greatly (e.g. bovine (approx. 18 mm); mouse (approx. 2 mm)) and confirms that mammalian lenses scale with size. The validated model includes two parameters: ß/α, which is the ratio of the proliferation rate in the peripheral and in the central region of the lens; and γ(GZ), a dimensionless pull-through parameter that accounts for the cell transition and exit from the epithelium into the lens body. Best-fit values were determined for mouse, rat, rabbit, bovine and human lens epithelia. The OPT model accounts for the peak in cell density at the periphery of the lens epithelium, a region where cell proliferation is concentrated and reaches a maximum coincident with the germinative zone. The ß/α ratio correlates with the measured FGF-2 gradient, a morphogen critical to lens cell survival, proliferation and differentiation. As proliferation declines with age, the OPT model predicted age-dependent changes in cell-density profiles, which we observed in mouse and human lenses.

3D adaptive optics in a light sheet microscope.

We report on a single plane illumination microscope (SPIM) incorporating adaptive optics in the imaging arm. We show how aberrations can occur from the sample mounting tube and quantify the aberrations both experimentally and computationally. A wavefront sensorless approach was taken to imaging a green fluorescent protein (GFP) labelled transgenic zebrafish. We show improvements in image quality whilst recording a 3D "z-stack" and show how the aberrations come from varying depths in the fish.

Use of fiber optic technology to measure the effects of anesthesia on luciferase reaction kinetics.

In vivo bioluminescent imaging (BLI) is a sensitive and reliable technique for studying gene expression, although experiments must be controlled tightly to obtain reproducible and quantitative measurements. The luciferase reaction depends on the availability of the reaction substrate, oxygen, and ATP, the distribution of which can vary markedly in different tissues. Here we used in vivo fiber optic technology, combined with stereotaxis-assisted surgery, to assess luciferase reaction kinetics in response to 2 anesthetic regimens, isoflurane and ketamine-xylazine. Transgenic rats that expressed luciferase under the control of the human prolactin promoter were used as a model organism. Anesthesia had a marked effect on luciferase reaction kinetics. The rise time to peak emission differed by 20 min between isoflurane and ketamine-xylazine. Optical imaging using a charge-coupled-device camera confirmed this delay. These results demonstrate that different anesthetics can have substantial effects on luciferase reaction kinetics and suggest that the timing of image acquisition after substrate injection should be optimized in regard to experimental conditions and the tissues of interest.

Real-time optical gating for three-dimensional beating heart imaging.

We demonstrate real-time microscope image gating to an arbitrary position in the cycle of the beating heart of a zebrafish embryo. We show how this can be used for high-precision prospective gating of fluorescence image slices of the moving heart. We also present initial results demonstrating the application of this technique to 3-D structural imaging of the beating embryonic heart.

Multidepth, multiparticle tracking for active microrheology using a smart camera.

The quantitative measurement of particle motion in optical tweezers is an important tool in the study of microrheology and can be used in a variety of scientific and industrial applications. Active microheology, in which the response of optically trapped particles to external driving forces is measured, is particularly useful in probing nonlinear viscoelastic behavior in complex fluids. Currently such experiments typically require independent measurements of the driving force and the trapped particle's response to be carefully synchronized, and therefore the experiments normally require analog equipment. In this paper we describe both a specialized camera and an imaging technique which make high-speed video microscopy a suitable tool for performing such measurements, without the need for separate measurement systems and synchronization. The use of a high-speed tracking camera based on a field programmable gate array to simultaneously track multiple particles is reported. By using this camera to simultaneously track one microsphere fixed to the wall of a driven sample chamber and another held in an optical trap, we demonstrate simultaneous optical measurement of the driving motion and the trapped probe particle response using a single instrument. Our technique is verified experimentally by active viscosity measurements on water-ethylene glycol mixtures using a phase-shift technique.