Biomedical subjects

Mark A Haidekker

Publications and source records attributed to Mark A Haidekker.

15 recordsLinked to original sources

PubMed2006

Optical fiber-based fluorescent viscosity sensor.

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.

Biosensing Techniques↗

PubMed2006

Influence of gold nanoparticles on collagen fibril morphology quantified using transmission electron microscopy and image analysis.

BACKGROUND: Development of implantable biosensors for disease detection is challenging because of poor biocompatibility of synthetic materials. A possible solution involves engineering interface materials that promote selfassembly and adhesion of autologous cells on sensor surfaces. Crosslinked type-I collagen is an acceptable material for developing engineered basement membranes. In this study, we used functionalized gold nanoparticles as the crosslinking agent. Functionalized nanoparticles provide sites for crosslinking collagen as well as sites to deliver signaling compounds that direct selfassembly and reduce inflammation. The goal of this study was to obtain a quantitative parameter to objectively determine the presence of crosslinks. METHODS: We analyzed TEM images of collagen fibrils by two methods: Run length analysis and topology analysis after medial axis transform. RESULTS: Run length analysis showed a significant reduction of the interfibril spaces in the presence of nanoparticles (change of 40%, P < 0.05), whereas the fibril thickness remained unchanged. In the topological network, the number of elements, number of branches and number of sides increased significantly in the presence of nanoparticles (P < 0.05). Other parameters, especially the number of loops showed only a minimal and nonsignificant change. We chose a ratiometric parameter of the number of branches normalized by the number of loops to achieve independence from gross fibril density. This parameter is lower by a factor of 2.8 in the presence of nanoparticles (P < 0.05). CONCLUSION: The numerical parameters presented herein allow not only to quantify fibril mesh complexity and crosslinking, but also to help quantitatively compare cell growth and adhesion on collagen matrices of different degree of crosslinking in further studies.

Journal Article↗

PubMed2006

A ratiometric fluorescent viscosity sensor.

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.

Acrylates↗

PubMed2005

Optical transillumination tomography with tolerance against refraction mismatch.

Optical transillumination tomography (OT) is a laser-based imaging modality where ballistic photons are used for projection generation. Image reconstruction is therefore similar to X-ray computed tomography. This modality promises fast image acquisition, good resolution and contrast, and inexpensive instrumentation for imaging of weakly scattering objects, such as for example tissue-engineered constructs. In spite of its advantages, OT is not widely used. One reason is its sensitivity towards changes in material refractive index along the light path. Beam refraction artefacts cause areas of overestimated tissue density and blur geometric details. A spatial filter, introduced into the beam path to eliminate scattered photons, will also remove refracted photons from the projections. In the projections, zones affected by refraction can be detected by thresholding. By using algebraic reconstruction techniques (ART) in conjunction with suitable interpolation algorithms, reconstruction artefacts can be partly avoided. Reconstructions from a test image were performed. Standard filtered backprojection (FBP) showed a round mean square (RMS) deviation from the original image of 9.9. RMS deviation with refraction-tolerant ART reconstruction was 0.33 and 0.24, depending on the algorithm, compared to 0.57 (FBP) and 0.06 (ART) in a non-refracting case. In addition, modified ART reconstruction allowed detection of small geometric details that were invisible in standard reconstructions. Refraction-tolerant ART may be the key to eliminating one of the major challenges of OT.

Algorithms↗

PubMed2005

Mercury free operation of the Coulter counter MultiSizer II sampling stand.

Electronic particle counters have gained widespread acceptance as a means to measure osmotic properties of cell membranes. Because most current instruments do not allow for the collection of true volume as a function of time data, investigators use older models such as the MultiSizer II sampling stand. A significant drawback to this and other older models is that they rely on mercury to maintain a constant pressure and to connect electrodes. The presence of mercury is a human health hazard that is exacerbated by the sometimes irregular vacuum pressures that cause mercury spills inside of the machine. To eliminate this hazard, we have determined that the MultiSizer II model can be simply and easily modified to function and collect temporal volume data without the use of mercury.

Animals↗

PubMed2005

Transillumination optical tomography of tissue-engineered blood vessels: a Monte Carlo simulation.

A Monte Carlo technique has been developed to simulate the transillumination laser computed tomography of tissue-engineered blood vessels. The blood vessel was modeled as a single cylinder layer mounted on a tubular mandrel. Sequences of images were acquired while rotating the mandrel. The tomographic image was reconstructed by applying a standard Radon transform. Angular discrimination was applied to simulate a spatial filter, which was used to reject multiply scattered photons. The simulation results indicated that the scattering effect can be overcome with angular discrimination because of the thin tissue thickness. However, any refractive-index mismatch among the tissue, the surrounding media, and the mandrel could produce significant distortions in the reconstructed image.

Animals↗

PubMed2005

Optical transillumination tomography for imaging of tissue-engineered blood vessels.

Recent progress in tissue engineering led to the development of completely biological human vessels grown from the patient's 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 microm and allowedto 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.

Animals↗

PubMed2005

Precision assessment of biofluid viscosity measurements using molecular rotors.

Blood viscosity changes with many pathologic conditions, but its importance has not been fully investigated because the current methods of measurement are poorly suited for clinical applications. The use of viscosity-sensitive fluorescent molecular rotors to determine fluid viscosity in a nonmechanical manner has been investigated recently, but it is unknown how the precision of the fluorescence-based method compares to established mechanical viscometry. Human blood plasma viscosity was modulated with high-viscosity plasma expanders, dextran, pentastarch, and hetastarch. The samples were divided into a calibration and a test set. The relationship between fluorescence emission and viscosity was established using the calibration set. Viscosity of the test set was determined by fluorescence and by cone-and-plate viscometer, and the precision of both methods compared. Molecular rotor fluorescence intensity showed a power law relationship with solution viscosity. Mechanical measurements deviated from the theoretical viscosity value by less than 7.6%, while fluorescence-based measurements deviated by less than 6%. The average coefficient of variation was 6.9% (mechanical measurement) and 3.4% to 3.8% (fluorescence-based measurement, depending on the molecular rotor used). Fluorescence-based viscometry exhibits comparable precision to mechanical viscometry. Fluorescence viscometry does not apply shear and is therefore more practical for biofluids which have apparent non-Newtonian properties. In addition, fluorescence instrumentation makes very fast serial measurements possible, thus promising new areas of application in laboratory and clinical settings.

Blood Chemical Analysis↗

PubMed2005

Interaction of fluorescent molecular rotors with blood plasma proteins.

Many disease states have associated blood viscosity changes. Molecular rotors, fluorescent molecules with viscosity sensitive quantum yields, have recently been investigated as a new method for biofluid viscosity measurement. Current viscometer measurements are complicated by proteins adhering to surfaces and forming air-surface layers. It is unknown at this time what effects proteins may have on biofluid viscosity measurements using molecular rotors. To answer this question, binding affinities to blood plasma proteins were investigated by equilibrium dialysis for four hydrophilic molecular rotors. Aqueous solutions of 9-[(2-cyano-2-hydroxy-carbonyl)vinyl]julolidine (CCVJ) and three derivatives were prepared and dialyzed against solutions of bovine source albumin, fibrinogen and immunoglobulin G approximating normal physiologic concentrations and fresh-frozen human plasma. After equilibration, dye concentration on each side of the dialysis membrane was assessed by spectrophotometry. The relative binding affinity of the four dyes to the proteins and to the plasma was compared. Affinity of all dyes was highest for albumin. The bound dye fraction showed little change in relation to protein concentration in the physiological concentration range. Diol, the most hydrophilic molecular rotor tested showed the lowest affinity for albumin. This study indicates that hydrophilic molecular rotors are well-suited for biofluid viscosity measurement.

Animals↗

PubMed2004

Extracellular signal-regulated kinase activation and endothelin-1 production in human endothelial cells exposed to vibration.

Hand-arm vibration syndrome is a vascular disease of occupational origin and a form of secondary Raynaud's phenomenon. Chronic exposure to hand-held vibrating tools may cause endothelial injury. This study investigates the biomechanical forces involved in the transduction of fluid vibration in the endothelium. Human endothelial cells were exposed to direct vibration and rapid low-volume fluid oscillation. Rapid low-volume fluid oscillation was used to simulate the effects of vibration by generating defined temporal gradients in fluid shear stress across an endothelial monolayer. Extracellular signal-regulated kinase (ERK1/2) phosphorylation and endothelin-1 (ET-1) release were monitored as specific biochemical markers for temporal gradients and endothelial response, respectively. Both vibrational methods were found to phosphorylate ERK1/2 in a similar pattern. At a fixed frequency of fluid oscillation where the duration of each pulse cycle remained constant, ERK1/2 phosphorylation increased with the increasing magnitude of the applied temporal gradient. However, when the frequency of flow oscillation was increased (thus decreasing the duration of each pulse cycle), ERK1/2 phosphorylation was attenuated across all temporal gradient flow profiles. Fluid oscillation significantly stimulated ET-1 release compared to steady flow, and endothelin-1 was also attenuated with the increase in oscillation frequency. Taken together, these results show that both the absolute magnitude of the temporal gradient and the frequency/duration of each pulse cycle play a role in the biomechanical transduction of fluid vibrational forces in endothelial cells. Furthermore, this study reports for the first time a link between the ERK1/2 signal transduction pathway and transmission of vibrational forces in the endothelium.

Blotting, Western↗

PubMed2004

Hydrophilic molecular rotor derivatives-synthesis and characterization.

Recent research shows high potential for some p-N,N-dialkylaminobenzylidenecyanoacetates, part of a group known as fluorescent molecular rotors, to serve as fluorescent, non-mechanical viscosity sensors. Of particular interest are molecules compatible with aqueous environments. In this study, we present the synthesis and physical characterization of derivatives from 9-(2-carboxy-2-cyanovinyl)-julolidine and related molecules. All compounds show a power-law relationship of fluorescence emission with the viscosity of the solvent, different mixtures of ethylene glycol and glycerol to modulate viscosity. Compounds with high water solubility exhibit the same behavior in aqueous solutions of dextran, where the dextran concentration was varied to modulate viscosity. In addition, some compounds have been found to have low sensitivity towards changes in the pH in the physiological range. The compounds presented show promise to be used in biofluids, such as blood plasma or lymphatic fluid, to rapidly and non-mechanically determine viscosity.

Acetates↗

PubMed2004

Computerized methods for X-ray-based small bone densitometry.

Animal models have been widely used to correlate in vivo changes in bone mineral density (BMD) with changes in disease state of bone. In small animal models, e.g. the hindlimb suspension model of bone loss, a non-invasive assessment of BMD is required. X-ray radiography has been surpassed in some cases by quantitative computed tomography (QCT) and dual X-ray absorptiometry (DEXA) quantitation. However, there are drawbacks in using the computerized methods, especially for small animals. In this paper, we present image-processing algorithms to quantitatively determine bone area and mineral density in digitized radiographs. Image calibration is based on a calibration step wedge, and the algorithm automatically detects the steps and computes the calibration data. In addition, we demonstrate how the algorithm can accurately determine the cortical outline of the bone and provide reliable data and statistics for small animal studies. A downloadable implementation example for the popular NIH Image package is provided.

Absorptiometry, Photon↗

PubMed2004

Cell membrane fluidity changes and membrane undulations observed using a laser scattering technique.

Local transversal micromotions of cell membranes have been reported. These may provide the physical basis for changes in membrane fluidity. In this study, tissue scattering properties are used to measure the magnitude of transversal micromotions under varying stimuli. Laser light is directed at the cell surface at a low incident angle of 12 degrees so that reflected or refracted light does not enter the microscope objective. Scattered light showed strong low-frequency intensity fluctuations with random-walk behavior. Fluctuations were quantified by the median coefficient of variation over the entire observed cell area. Incubation of the cells with protein crosslinkers (paraformaldehyde) and metabolic suppressors (sodium azide) suppressed these fluctuations by 62 and 44%, respectively. The application of hypoosmotic media caused an increase of fluctuation magnitude by 23% (p < 0.005). Agents that increase membrane fluidity (2% ethanol and xenon) increased fluctuation magnitude by 15 and 31%, respectively (p < 0.05). Cessation of the ethanol and xenon exposure led to partial recovery of the fluctuation magnitude, which was nonsignificant for ethanol. This study shows a strong link between membrane fluidity and transversal membrane undulations and provides an important step in the understanding of the mechanosensing function of the cell membrane.

Cell Line, Tumor↗

PubMed2002

Phospholipid-bound molecular rotors: synthesis and characterization.

Molecular rotors are fluorescent molecules with a viscosity-sensitive quantum yield that are often used to measure viscosity changes in cell membranes and liposomes. However, commercially available molecular rotors, such as DCVJ (1) do not localize in cell membranes but rapidly migrate into the cytoplasm leading to unreliable measurements of cell membrane viscosity. To overcome this problem, we synthesized molecular rotors covalently attached to a phospholipid scaffold. Attaching the rotor group to the hydrophobic end of phosphatidylcholine (PC) did not affect the rotor's viscosity sensitivity and allowed adequate integration into artificial bilayers as well as complete localization in the plasma membrane of an endothelial cell line. Moreover, these new rotors enabled the monitoring of phospholipid transition temperature. However, attachment of the rotor groups to the hydrophilic head of the phospholipid led to a partial loss of viscosity sensitivity. The improved sensitivity and exclusive localization in the cell plasma membrane exhibited by the phospholipid-bound molecular rotors suggest that these probes can be used for the study of membrane microviscosity.

Cell Line↗

PubMed2002

A novel approach to blood plasma viscosity measurement using fluorescent molecular rotors.

Molecular rotors, a group of fluorescent molecules with viscosity-dependent quantum yield, were tested for their suitability to act as fluorescence-based plasma viscometers. The viscosity of samples of human plasma was modified by the addition of pentastarch (molecular mass 260 kDa, 10% solution in saline) and measured with a Brookfield viscometer. Plasma viscosity was 1.6 mPa x s, and the mixtures ranged up to 4.5 mPa x s (21 degrees C). The stimulated light emission of the molecular rotors mixed in the plasma samples yielded light intensity that was nonoverlapping and of significantly different intensity for viscosity steps down to 0.3 mPa x s (n = 5, P < 0.0001). The mathematical relationship between intensity (I) and viscosity (eta) was found to be eta = (kappaI)(nu). After calibration and scaling the fluorescence based measurement had an average deviation versus the conventional viscometric measurements that was <1.8%. These results show the suitability of molecular rotors for fast, low-volume biofluid viscosity measurements achieving accuracy and precision comparable to mechanical viscometers.

Blood Viscosity↗