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ABSTRACT: BACKGROUND: Stink bugs represent a major agricultural pest complex attacking more than 200 wild and cultivated plants, including cotton in the southeastern US. Stink bug feeding on developing cotton bolls will cause boll abortion or lint staining and thus reduced yield and lint value. Current methods for stink bug detection involve manual harvesting and cracking open of a sizable number of immature cotton bolls for visual inspection. This process is cumbersome, time consuming, and requires a moderate level of experience to obtain accurate estimates. To improve detection of stink bug feeding, we present here a method based on fluorescent imaging and subsequent image analyses to determine the likelihood of stink bug damage in cotton bolls. RESULTS: Damage to different structures of cotton bolls including lint and carpal wall can be observed under blue LED-induced fluorescence. Generally speaking, damaged regions fluoresce green, whereas non-damaged regions with chlorophyll fluoresce red. However, similar fluorescence emission is also observable on cotton bolls that have not been fed upon by stink bugs. Criteria based on fluorescent intensity and the size of the fluorescent spot allow to differentiate between true positives (fluorescent regions associated with stink bug feeding) and false positives (fluorescent regions due to other causes). We found a detection rates with two combined criteria of 87% for true-positive marks and of 8% for false-positive marks. CONCLUSIONS: The imaging technique presented herein gives rise to a possible detection apparatus where a cotton boll is imaged in the field and images processed by software. The unique fluorescent signature left by stink bugs can be used to determine with high probability if a cotton boll has been punctured by a stink bug. We believe this technique, when integrated in a suitable device, could be used for more accurate detection in the field and allow for more optimized application of pest control.
Journal of Biological Engineering 02/2013; 7(1):5.
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ABSTRACT: We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity sensor). Characterization of their fluorescence properties was made with separate excitation of the units and through resonance energy transfer from the reference to the sensor dye. We have modified the linker and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm.
Bioorganic & medicinal chemistry 05/2012; 20(14):4443-50. · 2.82 Impact Factor
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ABSTRACT: Recent progress in tissue engineering led to the development of completely biological human vessels grown from the patients own cells. Those tissue-engineered blood vessels (TEBV) are grown on an individual basis at high costs per item, and therefore require close growth monitoring and quality control. We designed and tested an optical transillumination tomography system using red laser light to image weakly scattering specimens, such as TEBV. Fixated TEBV were imaged and the results compared to optical coherence tomography. This preliminary scanner prototype had an in-plane resolution of 50 m and allowed to see small inhomogeneities and defects in the samples. Tissue attenuation was found to be 70 cm–1. Main advantages of the transillumination tomography scanner over optical coherence tomography were the inexpensive instrumentation and the potential to rapidly acquire complete 3D sections with a CCD camera. The prototype presented in this study provides a basis to further improve image quality and acquisition speed.
Annals of Biomedical Engineering 04/2012; 33(3):323-327. · 2.37 Impact Factor
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ABSTRACT: Molecular rotors are a group of environment-sensitive fluorescent probes whose quantum yield depends on the ability to form twisted intramolecular charge-transfer (TICT) states. TICT formation is dominantly governed by the solvent's microviscosity, but polarity and the ability of the solvent to form hydrogen bonds play an additional role. The relationship between quantum yield ϕ(F) and viscosity η is widely accepted as a power-law, ϕ(F) = C · η(x). In this study, we isolated the direct influence of the temperature on the TICT formation rate by examining several molecular rotors in protic and aprotic solvents over a range of temperatures. Each solvent's viscosity was determined as a function of temperature and used in the above power-law to determine how the proportionality constant C varies with temperature. We found that the power-law relationship fully explains the variations of the measured steady-state intensity by temperature-induced variations of the solvent viscosity, and C can be assumed to be temperature-independent. The exponent x, however, was found to be significantly higher in aprotic solvents than in protic solvents. We conclude that the ability of the solvent to form hydrogen bonds has a major influence on the relationship between viscosity and quantum yield. To use molecular rotors for the quantitative determination of viscosity or microviscosity, the exponent x needs to be determined for each dye-solvent combination.
Journal of Fluorescence 09/2011; 22(1):457-65. · 2.11 Impact Factor
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ABSTRACT: Stink bugs (Hemiptera: Pentatomidae) comprise a critically important insect pest complex affecting 12 major crops worldwide including cotton. In the US, stink bug damage to developing cotton bolls causes boll abscission, lint staining, reduced fiber quality, and reduced yields with estimated losses ranging from 10 to 60 million dollars annually. Unfortunately, scouting for stink bug damage in the field is laborious and excessively time consuming. To improve scouting accuracy and efficiency, we investigated fluorescence changes in cotton boll tissues as a result of stink bug feeding.
Fluorescent imaging under long-wave ultraviolet light showed that stink bug-damaged lint, the inner carpal wall, and the outside of the boll emitted strong blue-green fluorescence in a circular region near the puncture wound, whereas undamaged tissue emissions occurred at different wavelengths; the much weaker emission of undamaged tissue was dominated by chlorophyll fluorescence. We further characterized the optimum emission and excitation spectra to distinguish between stink bug damaged bolls from undamaged bolls.
The observed characteristic fluorescence peaks associated with stink bug damage give rise to a fluorescence-based method to rapidly distinguish between undamaged and stink bug damaged cotton bolls. Based on the fluorescent fingerprint, we envision a fluorescence reflectance imaging or a fluorescence ratiometric device to assist pest management professionals with rapidly determining the extent of stink bug damage in a cotton field.
Journal of Biological Engineering 08/2011; 5(1):11.
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ABSTRACT: We describe the design, synthesis and fluorescent profile of a family of self-calibrating dyes that provide ratiometric measurements of fluid viscosity. The design is based on covalently linking a primary fluorophore (reference) that displays a viscosity-independent fluorescence emission with a secondary fluorophore (sensor) that exhibits a viscosity-sensitive fluorescence emission. Characterization of fluorescent properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. The chemical structures of both fluorophores and the linker length have been evaluated in order to optimize the overall brightness and sensitivity of the viscosity measurements. We also present an application of such ratiometric dyes for the detection of membrane viscosity changes in a liposome model.
Organic & Biomolecular Chemistry 03/2011; 9(9):3530-40. · 3.70 Impact Factor
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ABSTRACT: Molecular rotors are a form of fluorescent intramolecular charge-transfer complexes that can undergo intramolecular twisting motion upon photoexcitation. Twisted-state formation leads to non-radiative relaxation that competes with fluorescence emission. In bulk solutions, these molecules exhibit a viscosity-dependent quantum yield. On the molecular scale, the fluorescence emission is a function of the local free volume, which in turn is related to the local micro-viscosity. Membrane viscosity, and the inverse; fluidity, are characteristic terms used to describe the ease of movement withing the membrane. Often, changes in membrane viscosity govern intracellular processes and are indicative of a disease state. Molecular rotors have been used to investigate viscosity changes in liposomes and cells, but accuracy is affected by local concentration gradients and sample optical properties. We have developed self-calibrating ratiometric molecular rotors to overcome this challenge and integrated the new molecules into a DLPC liposome model exposed to the membrane-fluidizing agent propanol. We show that the ratiometric emission intensity linearly decreases with the propanol exposure and that the ratiometric intensity is widely independent of the total liposome concentration. Conversely, dye concentration inside liposomes influences the sensitivity of the system. We suggest that the new self-calibrating dyes can be used for real-time viscosity sensing in liposome systems with the advantages of lifetime measurements, but with low-cost steady-state instrumentation.
Biochimie 02/2011; 93(6):988-94. · 3.02 Impact Factor
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ABSTRACT: Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer states (TICT) upon photoexcitation. Some classes of molecular rotors, among them those that are built on the benzylidene malononitrile motif, return to the ground state either by nonradiative intramolecular rotation or by fluorescence emission. In low-viscosity solvents, intramolecular rotation dominates, and the fluorescence quantum yield is low. Higher solvent viscosities reduce the intramolecular rotation rate, thus increasing the quantum yield. We recently described a different mechanism whereby the fluorescence quantum yield of the molecular rotor also depends on the shear stress of the solvent. In this study, we examined a possible application for shear-sensitive molecular rotors for imaging flow patterns in fluidic chambers. Flow chambers with different geometries were constructed from polycarbonate or acrylic. Solutions of molecular rotors in ethylene glycol were injected into the chamber under controlled flow rates. LED-induced fluorescence (LIF) images of the flow chambers were taken with a digital camera, and the intensity difference between flow and no-flow images was visualized and compared to computed fluid dynamics (CFD) simulations. Intensity differences were detectable with average flow rates as low as 0.1 mm/s, and an exponential association between flow rate and intensity increase was found. Furthermore, a good qualitative match to computed fluid dynamics simulations was seen. On the other hand, prolonged exposure to light reduced the emission intensity. With its high sensitivity and high spatial and temporal resolution, imaging of flow patterns with molecular rotors may become a useful tool in microfluidics, flow measurement, and control.
Journal of Fluorescence 09/2010; 20(5):1087-98. · 2.11 Impact Factor
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ABSTRACT: It has been shown that compounds containing the p-N,N,-dialkylaminobenzylidene cyanoacetate motif can serve as fluorescent non-mechanical viscosity sensors. These compounds, referred to as molecular rotors, belong to a class of fluorescent probes that are known to form twisted intramolecular charge-transfer complexes in the excited state. In this study we present the synthesis and spectroscopic characterization of these compounds as viscosity sensors. The effects of the molecular structure and electronic density of these rotors to the emission wavelength, fluorescence intensity and viscosity sensitivity are discussed.
Tetrahedron 04/2010; 66(14):2582-2588. · 3.03 Impact Factor
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ABSTRACT: 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.
Journal of Biological Engineering 01/2010; 4:11.
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ABSTRACT: AbstractMolecular 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 steric hindrance, because it links the solvent's microviscosity to the formation rate
of 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 viscosity
measurement, probing dynamics of polymer formation, protein sensing and probing of protein aggregation, and microviscosity
probing in living cells.
Graphical Abstract
KeywordsBiofluids-Chemosensors-Emission spectroscopy-Mechanosensors-Optical properties-Polarity-Rheology-Twisted intramolecular charge transfer-Viscosity
12/2009: pages 267-308;
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ChemMedChem 12/2009; 5(1):56-60. · 3.15 Impact Factor
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ABSTRACT: In the non-amyloidogenic pathway, amyloid precursor protein (APP) is cleaved by alpha-secretases to produce alpha-secretase-cleaved soluble APP (sAPP(alpha)) with neuroprotective and neurotrophic properties; therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of Alzheimer's disease. Here, we demonstrate the effects of type III secretory phospholipase A(2) (sPLA(2)-III) on sAPP(alpha) secretion. Exposing differentiated neuronal cells (SH-SY5Y cells and primary rat neurons) to sPLA(2)-III for 24 h increased sAPP(alpha) secretion and decreased levels of Abeta(1-42) in SH-SY5Y cells, and these changes were accompanied by increased membrane fluidity. We further tested whether sPLA(2)-III-enhanced sAPP(alpha) release is due in part to the production of its hydrolyzed products, including arachidonic acid (AA), palmitic acid (PA), and lysophosphatidylcholine (LPC). Addition of AA but neither PA nor LPC mimicked sPLA(2)-III-induced increases in sAPP(alpha) secretion and membrane fluidity. Treatment with sPLA(2)-III and AA increased accumulation of APP at the cell surface but did not alter total expressions of APP, alpha-secretases, and beta-site APP cleaving enzyme. Taken together, these results support the hypothesis that sPLA(2)-III enhances sAPP(alpha) secretion through its action to increase membrane fluidity and recruitment of APP at the cell surface.
The Journal of Lipid Research 10/2009; 51(5):957-66. · 5.56 Impact Factor
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ABSTRACT: Techniques such as optical coherence tomography and diffuse optical tomography have been shown to effectively image highly scattering samples such as tissue. An additional modality has received much less attention: Optical transillumination (OT) tomography, a modality that promises very high acquisition speed for volumetric scans. With the motivation to image tissue-engineered blood vessels for possible biomechanical testing, we have developed a fast OT device using a collimated, noncoherent beam with a large diameter together with a large-size CMOS camera that has the ability to acquire 3D projections in a single revolution of the sample. In addition, we used accelerated iterative reconstruction techniques to improve image reconstruction speed, while at the same time obtaining better image quality than through filtered backprojection. The device was tested using ink-filled polytetrafluorethylene tubes to determine geometric reconstruction accuracy and recovery of absorbance. Even in the presence of minor refractive index mismatch, the weighted error of the measured radius was <5% in all cases, and a high linear correlation of ink absorbance determined with a photospectrometer of R(2) = 0.99 was found, although the OT device systematically underestimated absorbance. Reconstruction time was improved from several hours (standard arithmetic reconstruction) to 90 s per slice with our optimized algorithm. Composed of only a light source, two spatial filters, a sample bath, and a CMOS camera, this device was extremely simple and cost-efficient to build.
Annals of biomedical engineering 10/2008; 36(10):1699-707. · 2.41 Impact Factor
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ABSTRACT: 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.
Biochimica et Biophysica Acta 05/2008; 1778(4):1148-53. · 4.66 Impact Factor
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ABSTRACT: 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 solvent's 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.
Organic & Biomolecular Chemistry 07/2007; 5(11):1669-78. · 3.70 Impact Factor
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ABSTRACT: 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 lambda(ex) = 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.
Nature Protocol 01/2007; 2(1):227-36. · 8.36 Impact Factor
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ABSTRACT: Molecular rotors are a unique group of viscosity-sensitive fluorescent probes. Several recent studies have shown their applicability as nonmechanical fluid viscosity sensors, particularly in biofluids containing proteins. To date, molecular rotors have had to be dissolved in the fluid for the measurement to be taken. We now show that molecular rotors may be covalently bound to a fiber-optic tip without loss of viscosity sensitivity. The optical fiber itself may be used as a light guide for emission light (external illumination of the tip) as well as for both emission and excitation light. Covalently bound molecular rotors exhibit a viscosity-dependent intensity increase similar to molecular rotors in solution. An optical fiber-based fluorescent viscosity sensor may be used in real-time measurement applications ranging from biomedical applications to the food industry.
Optics Letters 10/2006; 31(17):2529-31. · 3.40 Impact Factor
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ABSTRACT: We compared the ability of two different sensors to probe the polymerization dynamics of three popular biopolymers: Polyacrylamide, type-I collagen, and tetramethoxysilane-based sol-gel. The sensors were (1) a magnetoelastic (ME) strip of amorphous metal-glass material performing a viscosity-dependent dampened oscillation after excitation in a radiofrequency magnetic field, and (2) a fluorescent molecular rotor with a viscosity-dependent quantum yield. Both the resonance quality factor Q of the ME strip and the emission intensity of the molecular rotor, exposed to the polymers, were recorded after initiating the polymerization reaction. In polyacrylamide, the Q factor of the ME strip decreased in an exponential fashion, reflecting increased rigidity of the progressively crosslinking monomers. The molecular rotor was quenched by the free-radical donor ammonium persulfate. In collagen gels, increasing crosslinking was accompanied by a marked increase of molecular rotor emission intensity. In contrast, the ME strip showed only a minor decrease in Q. Sol-gel polymerization was accompanied by a decrease of Q of the ME strip. Molecular rotor intensity exhibited a more complex pattern, where hydrolysis and polymerization phases could be distinguished. In conclusion, both the ME strip and the molecular rotors allow monitoring of polymerization processes in real-time. Fluorescent probing of the collagen gelation process and final rigidity is therefore feasible. Finally, both sensors showed a strong reaction in sol-gels, but also appeared to provide information on the dynamics of the gelation process, namely the hydrolysis and crosslinking phase.
Sensor Letters 08/2006; 4(3):257-261. · 0.82 Impact Factor
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ABSTRACT: The development of a dual probe that provides ratiometric measurements of fluid viscosity is described. The design is based on coupling of a primary fluorophore with viscosity-independent fluorescence emission (blue unit) with a secondary fluorophore that exhibits viscosity-sensitive fluorescent emission quantum yield (red unit). Excitation of the secondary fluorophore can be achieved via Resonance Energy Transfer. The ratio of the fluorescence emission of these fluorophores provides an accurate, ratiometric measurement of solvent viscosity.
Journal of the American Chemical Society 02/2006; 128(2):398-9. · 9.91 Impact Factor
