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PubMed2006

Dual polarization interferometry characterization of carbohydrate-protein interactions.

Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. The technique was applied to the analysis of carbohydrate-protein interactions. The selected system involved a 12-kDa recombinant fragment of collagen V (HepV) and heparin, a complex polysaccharide. Here we report on the analysis of thickness, density, and mass of surface structures obtained during the binding of HepV to heparin, which is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. This system, which was initially studied for its biological relevance, displayed anomalous behavior in kinetic studies using surface plasmon resonance (SPR) assays that has been attributed to putative conformational changes. It was this putative conformational change that prompted us to investigate the binding using an alternative analytical approach. While using DPI to monitor binding events, a streptavidin layer (surface coverage 2.105 ng mm(-2)) was bound to the sensor surface (92% coverage), which captured 0.105 ng mm(-2) of biotinylated heparin (a stoichiometric ratio of 1:6 heparin-streptavidin). The heparin inserted into the streptavidin layer but was still found to be capable of binding 0.154 ng mm(-2) of HepV, which was also observed to insert into the streptavidin layer. This allowed the reliable calculation of the stoichiometric ratio for the HepV-heparin complex ( approximately 1.7:1.0), which has proved to be difficult to evaluate by SPR assays. Furthermore, real-time analysis of the heparin-HepV interaction by DPI suggested that there was some surface loss (probably of streptavidin) while the binding was occurring rather than the putative conformational change that has been suggested on the basis of kinetic data alone. This gives further insight into the binding mechanism of HepV to heparin.

Binding Sites↗
PubMed2005

Dual polarization interferometry size and density characterisation of DNA immobilisation and hybridisation.

Investigation of nucleic acid interactions was performed using dual polarization interferometry, a novel approach to elucidating molecular interactions. This paper presents a preliminary study of adsorption of single stranded DNA onto functionalised silicon oxynitride, compared with covalent linkage, and avidin-biotin immobilisation. The effect of probe concentration on hybridisation efficiency was also examined. We found that increasing the electrolyte concentration resulted in a decrease of adsorbed DNA and that capture of a biotinylated duplex DNA on an adsorbed avidin layer resulted in four times fewer molecules per cm(2) than for duplex DNA covalently bound via an amine end terminal. The rate of thickness increase of a biotin probe layer on an adsorbed avidin capture layer increased 10-fold when the probe concentration was increased from 0.1 microM to 1 microM. The close grafting density of the higher concentration probe meant that the immobilised probes were unavailable for hybridisation.

Biosensing Techniques↗
PubMed2005

Dual polarisation interferometry characterisation of DNA immobilisation and hybridisation detection on a silanised support.

Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterise the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19 mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was approximately 35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was approximately 85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.

Biosensing Techniques↗
PubMed2006

A comparison between dual polarization interferometry (DPI) and surface plasmon resonance (SPR) for protein adsorption studies.

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30-1.35 mg/m(2) at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.

Adsorption↗
PubMed2004

Non-destructive characterization of resin-based filling materials using Electronic Speckle Pattern Interferometry.

OBJECTIVES: The aim of this study was to develop a non-destructive test for characterizing the modulus of resin-based filling materials. METHODS: Five different visible-light-cured composites (Filtek A110 [AO], Z100 [ZO], Filtek Z250 [ZT], F2000 [FT] and Filtek Flow [FF]; 3M-ESPE) were selected for this study. An ESPI (Electronic Speckle Pattern Interferometry) apparatus involving cantilever beam specimens (28 mm long, 8 mm wide and 2 mm thick) was developed for determining modulus. Out-of-plane displacement upon force application was measured using two focus region lengths (22 mm [EI1]; 11 mm [EI2]) and modulus (n = 7) was subsequently computed based on cantilever beam equations. Data was compared to those obtained from three-point-bend flexural testing (n = 7) based on ISO4049:2000 specifications [ISO]. Specimens were stored in distilled water at 37 degrees C for 1 week prior to evaluation for all tests. Results were analyzed using ANOVA/Scheffe's post-hoc tests (p < 0.05) and Pearson's correlation (p < 0.01). RESULTS: Modulus ranged from 5.53 to 13.99, 5.78 to 14.24 and 4.26 to 11.30 GPa for EI1, EI2 and ISO, respectively. For all three tests, the modulus of ZO and FT was significantly greater than ZT, which in turn was significantly greater than AO and FF. Correlation of EI1 and EI2 to ISO was significant, positive and very strong (r = 0.94 for EI1 and EI2). SIGNIFICANCE: ESPI may be a viable method of characterizing the modulus of resin-based filling materials. As it is a non-destructive test, time-dependent effects of composites can be determined using the same specimens leading to substantial time and material savings.

Analysis of Variance↗
PubMed2006

Low coherence interferometry of the cochlear partition.

Interferometric measurement of the vibration of the organ of Corti in the isolated guinea pig cochlea was conducted using low-coherence light (1310+/-47 nm wavelength) from a superluminescent diode. The short coherence length of the light source localized measurements along the axial direction to within a approximately 10-microm window (in tissue), even when using a low numerical-aperture lens. The ability to accomplish this is important because measurement of the vibration of the basal-turn organ of Corti is generally done via a small hole in the bone of the cochlea, which effectively limits the numerical aperture. The axial localization, combined with the inherent sensitivity of the method, allowed distinct measurements of the basilar membrane (BM) and the putative reticular lamina (RL) vibration using only the native tissue reflectance, that is without requiring the use of reflective particles. The system was first operated in a scanning mode as an optical coherence tomography (OCT) system to yield an image of the organ of Corti. The reflectance of intensity from the BM and RL was 8x10(-5) and 8x10(-6), respectively. The internal structure between the BM and RL presented a variable reflectivity of about 10(-7). A mirror would define a reflectance of 1.00. Then the instrument was operated as a homodyne interferometer to measure the displacement of either the BM or RL. Vibration at 16 kHz was induced by a piezoelectric actuator, causing whole movement of a dissected cochlea. After calibration of the system, we demonstrated clear measurement of mechanically driven vibration for both the BM and RL of 0.30 nm above a noise floor equivalent to 0.03 nm. OCT interferometry, when adapted for in vivo organ of Corti measurements, appears suitable to determine the micromechanical vibration of cells and tissue elements of the organ.

Animals↗
PubMed2006

Anisotropic Poisson's ratio and compression modulus of cortical bone determined by speckle interferometry.

Young's modulus and Poisson's ratios of 6mm-sized cubes of equine cortical bone were measured in compression using a micro-mechanical loading device. Surface displacements were determined by electronic speckle pattern-correlation interferometry. This method allows for non-destructive testing of very small samples in water. Analyses of standard materials showed that the method is accurate and precise for determining both Young's modulus and Poisson's ratio. Material properties were determined concurrently in three orthogonal anatomic directions (axial, radial and transverse). Young's modulus values were found to be anisotropic and consistent with values of equine cortical bone reported in the literature. Poisson's ratios were also found to be anisotropic, but lower than those previously reported. Poisson's ratios for the radial-transverse and transverse-radial directions were 0.15+/-0.02, for the axial-transverse and axial-radial directions 0.19+/-0.04, and for the transverse-axial and radial-axial direction 0.09+/-0.02 (mean+/-SD). Cubes located only millimetres apart had significantly different elastic properties, showing that significant spatial variation occurs in equine cortical bone.

Animals↗
PubMed2006

Age-related behavior of posterior chamber lenses in myopic phakic eyes during accommodation measured by anterior segment partial coherence interferometry.

PURPOSE: To evaluate age-related position shifts of the crystalline lens and the implantable contact lens (ICL, Staar Surgical) by a new, commercially available, anterior segment partial coherence interferometer, the ACMaster (Carl Zeiss Meditec), during accommodation in myopic eyes. SETTING: ALZ Augenklinik, Munich, Germany. METHODS: Fifty-three eyes of 29 consecutive patients were measured after myopic ICL implantation before and during subjective accommodation to a stimulus of 3 diopters (D) by anterior segment partial coherence interferometry (PCI). Nine eyes were also measured with a 5.00 diopters (D) stimulus; 14 eyes were measured repeatedly at different visits. The mean patient age was 33 years +/- 9 (SD) (range 21 to 59 years). The preoperative mean sphere was -7.6 +/- 1.9 D (range -5.0 to -11.5 D) and the cylinder, -1.4 +/- 1.1 D (range 0 to -4.25 D). RESULTS: Older patients had a tendency toward smaller vaults on desaccommodation between the ICL and the crystalline lens compared to younger individuals. In younger patients, there was a decrease of the vault on accommodation, whereas it increased in older persons (P = .005). During accommodation, the more the anterior lens surface shifted forward, the more the ICL bulged (P = .005). The change in vaulting was significantly larger at 5.00 D than at 3.00 D accommodation stimulus (P = .012). CONCLUSIONS: The behavior of ICLs in relation to the crystalline lens during accommodation varied with age and could be shown by PCI. The position shift of the ICL depended on the initial vault at desaccommodation and the ability of the anterior lens surface to bulge forward. Even though the crystalline lens stiffened, and therefore accommodation deteriorated with age, there was still a movement of the ICL, pointing to the role of the ciliary muscle movement in accommodation.

Accommodation, Ocular↗
PubMed2006

Pseudophakic accommodation and pseudoaccommodation under physiological conditions measured with partial coherence interferometry.

PURPOSE: To distinguish pseudophakic accommodation from pseudoaccommodation by measuring the physiologically and pharmacologically induced anterior chamber depth (ACD) shifts. SETTING: Department of Ophthalmology, University of Debrecen, Debrecen, Hungary. METHODS: This study comprised 100 pseudophakic eyes of 79 patients. Forty patients (Group 1) received the AcrySof MA60AC intraocular lens (IOL) (Alcon Laboratories), 50 patients (Group 2) received the SA60AT IOL (Alcon Laboratories), and 10 patients (Group 3) received the apodized diffractive SA60D3 ReSTOR IOL. Visual function was evaluated a mean of 10.2 months +/- 9.2 (SD) postoperatively, and the total pseudoaccommodative amplitude was determined with a defocusing technique. To distinguish pseudophakic accommodation from pseudoaccommodation, ACD measurements were performed using partial coherence interferometry during distance fixation and physiologic accommodation after pharmacologic relaxation of the ciliary muscle. RESULTS: Best corrected distance and near visual acuities were similar in the 3 groups (P = .75 and P = .08, respectively). Distance corrected near visual acuity was significantly better in Group 3 (P < .001), with all eyes achieving J1 or better. Three percent in Group 1 and 8% in Group 2 achieved J1 or better. Subjective accommodation was similar in Groups 1 and 2 (-0.82 +/- 0.18 diopter [D] and -1.00 +/- 0.35 D, respectively; P = .3). Group 3 had an accommodation curve with 2 peaks. Intraocular lens movement differences between the groups were not significant (physiologic stimulus: P = .07; cyclopentolate: P = .46), and significant ACD shifts from baseline were not detected (physiologic stimulus: P = .14; cyclopentolate: P = .10). CONCLUSIONS: Pseudoaccommodative amplitude of the investigated monofocal IOLs was independent of IOL movement. Anterior shift did not affect good near visual acuity with the AcrySof ReSTOR IOL.

Accommodation, Ocular↗
PubMed2004

In vivo biometry in the mouse eye with low coherence interferometry.

PURPOSE: A major drawback of the mouse model of myopia is that the ocular dimensions cannot be measured in vivo, and that histological techniques post-mortem suffer from limited resolution. We have tested the potential of a newly developed technique, optical low coherence interferometry (OLCI), adapted for short measurement distances by Meditec, Carl Zeiss, Jena, Germany (the "ACMaster"). Using this technique, ocular biometry was performed in mice with normal vision and after deprivation of form vision. METHODS: Axial eye length, corneal thickness and anterior chamber depth were measured in 23 mice, aged 25-53 days, and standard deviations from repeated measurements in the same eyes, as well as intra-individual and inter-individual variability were determined in different age groups. The data were compared to those from a preceding study in which biometrical data were obtained from frozen sections [Vision Res. 44 (2004) 1857]. Refractions were measured by automated infrared photorefraction. Mice had either normal visual exposure or were monocularly deprived of form vision for 14 days. RESULTS: Using OLCI, axial length could be determined with an average standard deviation of 8.0 +/- 2.9 microm, corneal thickness with 3.5 +/- 2.1 microm, and anterior chamber depth with 10.6 +/- 12.3 microm. Neither axial length, nor corneal thickness, nor anterior chamber depth were significantly different in left and right eyes of individual mice that had normal visual experience (mean absolute difference between axial lengths: 17 +/- 18 microm, between corneal thickness 5.1 +/- 4.8 microm, and between anterior chamber depths 16.7 +/- 14.8 microm). Compared to the variability that was previously found in frozen sections, the variability of axial length measurements with OLCI was 2.7 times less. After two weeks of form deprivation, OLCI revealed a significant axial elongation in the occluded eyes, compared to the contralateral fellow eyes (+38 +/- 36 microm or 1.16%, p = 0.045, n = 7, paired t-test). In this sample, no accompanying myopic shift was observed in the occluded eyes but this observation is not unexpected given the inherently variable responses of mouse eye growth to visual deprivation. CONCLUSION: OLCI had sufficient resolution in living mice to detect axial length changes in vivo that were equivalent to a dioptric change of 2 D. Using this technique, it was confirmed that mouse eyes respond to form deprivation by axial elongation, similar to the eyes of other animal models. The lack of a myopic shift in this sample, despite the axial elongation, demonstrates that biometric data are particularly important when the mouse eye is used as a model to study myopia.

Animals↗
PubMed2001

Hydration state of single cytochrome c monolayers on soft interfaces via neutron interferometry.

Yeast cytochrome c (YCC) can be covalently tethered to, and thereby vectorially oriented on, the soft surface of a mixed endgroup (e.g., -CH3/-SH = 6:1, or -OH/-SH = 6:1) organic self-assembled monolayer (SAM) chemisorbed on the surface of a silicon substrate utilizing a disulfide linkage between its unique surface cysteine residue and a thiol endgroup. Neutron reflectivities from such monolayers of YCC on Fe/Si or Fe/Au/Si multilayer substrates with H2O versus D2O hydrating the protein monolayer at 88% relative humidity for the nonpolar SAM (-CH3/-SH = 6:1 mixed endgroups) surface and 81% for the uncharged-polar SAM (-OH/-SH = 6:1mixed endgroups) surface were collected on the NG1 reflectometer at NIST. These data were analyzed using a new interferometric phasing method employing the neutron scattering contrast between the Si and Fe layers in a single reference multilayer structure and a constrained refinement approach utilizing the finite extent of the gradient of the profile structures for the systems. This provided the water distribution profiles for the two tethered protein monolayers consistent with their electron density profile determined previously via x-ray interferometry (Chupa et al., 1994).

Biophysical Phenomena↗
PubMed2002

Cellular organization and substructure measured using angle-resolved low-coherence interferometry.

We measure the organization and substructure of HT29 epithelial cells in a monolayer using angle-resolved low-coherence interferometry. This new technique probes cellular structure by measuring scattered light, as in flow cytometry, but offers an advantage in that the structure can be examined in situ, avoiding the need to disrupt the cell monolayer. We determine the size distribution of the cell nuclei by fitting measured light-scattering spectra to the predictions of Mie theory. In addition, we obtain information about the cellular organization and substructure by examining the spatial correlations within the monolayer. A remarkable finding is that the spatial correlations over small length scales take the form of an inverse power law, indicating the fractal nature of the packing of the subcellular structures. We also identify spatial correlations on a scale large compared with the size of a cell, indicating an overlying order within the monolayer.

Biophysics↗
PubMedDate not supplied

Determination of particle sedimentation rate by ultrasonic interferometry: role of particle size, density and volume fraction.

The sedimentation rate (SR) of non-aggregated spherical particles in suspension was determined using an ultrasonic interferometry technique (Echo-Cell); this method is based on A-mode echography and measures the rate of formation of a sediment on a solid plate during settling. The particle accumulation rate, which is related to SR, is obtained from the interference of two waves reflected by two interfaces: one between the plate and the sediment and the other between the sediment and the suspension. Studies were carried out at 25 degrees C using latex spheres of different diameters (7 to 20 micron) and densities (1.062 to 1.190 g/cm3) suspended in distilled water at various volume fractions (1% to 5%). As anticipated by the Stokes model, linear relations were found between SR and both particle density and the square of particle radius. Experimental SR values decreased with increasing suspension particle concentration; these concentration effects were in good agreement with those predicted by the Steinour model. Our results thus serve to validate the theoretical aspects of the Echo-Cell method and suggest its usefulness as a tool for studies of RBC interaction and RBC aggregation.

Blood Sedimentation↗
PubMed1996

Measurement of human platelet microaggregates by a new method: ultrasonic interferometry.

We have adapted the ultrasonic interferometry technique (Echo-Cell), which was initially designed to study red blood cell aggregation and agglutination, to the detection of human platelet microaggregates. The experimental parameter chosen was the slope of the signal over the first 5 minutes of sedimentation. We compared our new method with the conventional aggregometry for the measurement of aggregates after thrombin-, collagen-, and epinephrine-induced platelet activation. Under these conditions we demonstrated the particular sensibility of the present method in detecting small platelet aggregates induced in the first phase of aggregation and formed by low concentrations of agonists. Furthermore, as an illustration of this method, we showed an inhibition of the formation of thrombin-induced platelet aggregates in a concentration-dependent manner by the well known antagonist arginine-glycine-aspartic acid-serine with a median inhibitory concentration of 0.4 micromol/L, which is 30 times lower than the median inhibitory concentration found by aggregometry.

Blood Platelets↗
PubMed1985

Efficacy of laser interferometry in predicting visual result of YAG laser posterior capsulotomy.

One hundred fifteen patients with cloudy posterior capsules that required capsulotomies were evaluated by laser interferometry. The predicted acuity was within one line of postoperative acuity in 44 patients (38.3%) and within two lines in 73 patients (63.5%). The correlation coefficient between the predicted and actual post-capsulotomy acuities was 0.38. However, the predicted retinal visual acuity was compared to postoperative Snellen visual acuity and found to differ by an average of three lines. A large number of false negative results reduced the clinical usefulness of the test.

Adolescent↗
PubMed2001

Analysis on the nature of thermally induced deformation in human dentine by electronic speckle pattern interferometry (ESPI).

OBJECTIVE: To examine the in-plane and out-of-plane response of human dentine to thermal loads in real time. METHODS: An Electronic Speckle Pattern Interferometry (ESPI) system sensitive to both the in-plane and out-of-plane displacements was configured and used in conjunction with an advanced fringe processing technique. Specimens were prepared from freshly extracted lower central incisor teeth and were separately mounted on a thermal block to apply thermal loads from room temperature (25 degrees C) to 60 degrees C. The real time speckle patterns were acquired using a digital camera. These digital fringe patterns were subjected to further image processing to enhance the quality of fringes. The resultant images were later analyzed to study the out-of-plane and in-plane displacement gradients in the facio-lingual plane of the dentine. RESULTS: The out-of-plane deformations were observed in the plane perpendicular to the long axis of the tooth, while the in-plane deformations occurred in the plane parallel to the long axis of the tooth. CONCLUSION: The ESPI analysis revealed whole-field and distinct thermal response in human dentine in-plane and out-of-plane. The cervical dentine experienced distinct and conspicuous displacement to the temperature changes.

Adult↗
PubMed2000

Diffusion of lysozyme in gels and liquids. A general approach for the determination of diffusion coefficients using holographic laser interferometry.

A study on diffusion measurements of the protein lysozyme in liquids and agarose gels, at different pH and ionic strengths, has been performed using holographic laser interferometry. The measurements showed that the diffusive flux was very dependent on pH and ionic strength when the protein was not at its isoelectric point or when the charge of the lysozyme molecules was not screened by ions in the solution. Evaluation of the experimental data with Fick's law, resulted in diffusion coefficients for lysozyme that are strongly dependent on pH and ionic strength. Evaluation of the experimental data using a more general transport model, based on chemical potential gradients instead of concentration gradients resulted in lysozyme diffusion coefficients that are independent of pH and ionic strength. The chemical potential was estimated by using the Poisson-Boltzmann equation.

Diffusion↗
PubMed2001

Reproducibility of optical biometry using partial coherence interferometry : intraobserver and interobserver reliability.

PURPOSE: To evaluate the intraobserver and interobserver variability in axial length (AL), anterior chamber depth (ACD), and corneal radius measurements using an optical biometry instrument based on partial coherence interferometry (PCI). SETTING: Johannes Gutenberg University, Mainz, Germany. METHODS: In this observational case series and interobserver reliability trial, 30 healthy, emmetropic to moderately myopic eyes of 15 volunteers were evaluated. The AL, ACD, and corneal radius were measured 20 times in 10 eyes by 1 observer to evaluate the intraobserver variability. To evaluate the interobserver variability, the measurements were taken in 20 eyes by 5 different observers. Measurements were performed using the IOLMaster (Carl Zeiss) based on PCI. The data description is based on coefficients of variation and the statistical inference on reliability estimation based on analysis of variance. The main outcome measures were intraobserver and interobserver variability and reliability in AL, ACD, and corneal radii. RESULTS: The intraobserver variability (SD) was +/-25.6 microm for AL, +/-33.4 microm for ACD, and +/-12.9 microm for corneal radius. The coefficients of variation were 0.1%, 0.9%, and 0.17%, respectively. The interobserver variability (SD) was +/-21.5 microm for AL, +/-29.8 microm for ACD, and +/-15.9 microm for corneal radius. The coefficients of variation were 0.09%, 0.82%, and 0.21%, respectively. The reliability was 99.9% for AL, 97.8% for ACD, and 99.8%/99.5% for corneal radius (r1/r2). The slightly reduced reliability in ACD measurement was caused by a reduced intraobserver reliability of 1 of the 5 observers (R = 0.87). CONCLUSION: Partial coherence biometry using the IOLMaster for AL measurement was highly reliable, offering observer-independent measurement results.

Adult↗