Goniometer for determination of the spectrally resolved scattering phase function of suspended particles.

An apparatus for direct determination of the spectral resolved scattering phase function of suspensions and emulsions is described. The system consists of a polychromatic xenon lamp as a light source and a spectrometer as a detector. Together with a stepper motor, the system enables spectrally and angularly resolved measurements in the range of 450 nm-950 nm and 10°-170°, respectively. A model for light propagation inside the cuvette in the regime of single scattering, which also takes the spectral dimension into account, was developed. A postprocessing algorithm applying the model and an extrapolation to the measured angular data allows a direct determination of the complete scattering phase function. By comparing measurements on polystyrene microspheres with Mie theory, the concept of the presented instrument was validated. Finally, the method was used to determine the scattering phase function of different types and brands of soybean oil emulsions such as Intralipid or Lipovenös. The measured scattering phase functions were then used to calculate the corresponding Legendre moments to an order of 20 and for wavelengths between 450 nm and 750 nm (available online), which besides the anisotropy factor also allow the determination of higher order factors such as gamma.

Online estimation of optimal operating conditions for simulated moving bed chromatographic processes.

A new approach for determining optimal operating conditions for simulated moving bed chromatographic processes is presented. The method is based on recursive online estimation and requires only rough initial estimates. It is based on a simple foot point model of the moving concentration fronts and an online measurement of the corresponding retention times in the different zones of the plant. A mathematical representation of the adsorption isotherms is not required. The method is validated experimentally for the separation of bicalutamide enantiomers.

Polarization influence on reflectance measurements in the spatial frequency domain.

In this work, we quantify the influence of crossed polarizers on reflectance measurements in the spatial frequency domain. The use of crossed polarizers is a very common approach for suppression of specular surface reflections. However, measurements are typically evaluated using a non-polarized scalar theory. The consequences of this discrepancy are the focus of our study, and we also quantify the related errors of the derived optical properties. We used polarized Monte Carlo simulations for forward calculation of the reflectance from different samples. The samples' scatterers are assumed to be spherical, allowing for the calculation of the scattering functions by Mie theory. From the forward calculations, the reduced scattering coefficient [Formula: see text] and the absorption coefficient µa were derived by means of a scalar theory, as commonly used. Here, we use the analytical solution of the scalar radiative transfer equation. With this evaluation approach, which does not consider polarization, we found large errors in [Formula: see text] and µa in the range of 25% and above. Furthermore, we investigated the applicability of the use of a reference measurement to reduce these errors as suggested in literature. We found that this method is not able to generally improve the accuracy of measurements in the spatial frequency domain. Our general recommendation is to apply a polarized theory when using crossed polarizers.

Light scattering microscopy with angular resolution and its possible application to apoptosis.

An inverted microscope has been modified for light scattering experiments with high angular resolution in combination with transmission, wide-field fluorescence or laser scanning microscopy. Supported by simulations of Mie scattering, this method permits detection of morphological changes of 3T3 fibroblasts on apoptosis and formation of spherically shaped cells of about 20 µm diameter, in agreement with visual observation. Smaller sub-structures (e.g. cell nuclei) as well as cell clusters may possibly contribute to the scattering behaviour. Results of 2-dimensional cell cultures are confirmed by 3-dimensional multicellular spheroids of 3T3 fibroblasts and HeLa 2E8 cervix carcinoma cells, where in most cases no morphological changes are discernable. This offers some advantage of light scattering microscopy for label-free detection of apoptosis and may represent a first step towards label-free in vivo diagnostics.

Determination of reference values for optical properties of liquid phantoms based on Intralipid and India ink.

A multi-center study has been set up to accurately characterize the optical properties of diffusive liquid phantoms based on Intralipid and India ink at near-infrared (NIR) wavelengths. Nine research laboratories from six countries adopting different measurement techniques, instrumental set-ups, and data analysis methods determined at their best the optical properties and relative uncertainties of diffusive dilutions prepared with common samples of the two compounds. By exploiting a suitable statistical model, comprehensive reference values at three NIR wavelengths for the intrinsic absorption coefficient of India ink and the intrinsic reduced scattering coefficient of Intralipid-20% were determined with an uncertainty of about 2% or better, depending on the wavelength considered, and 1%, respectively. Even if in this study we focused on particular batches of India ink and Intralipid, the reference values determined here represent a solid and useful starting point for preparing diffusive liquid phantoms with accurately defined optical properties. Furthermore, due to the ready availability, low cost, long-term stability and batch-to-batch reproducibility of these compounds, they provide a unique fundamental tool for the calibration and performance assessment of diffuse optical spectroscopy instrumentation intended to be used in laboratory or clinical environment. Finally, the collaborative work presented here demonstrates that the accuracy level attained in this work for optical properties of diffusive phantoms is reliable.

Multiple scattering of polarized light: influence of absorption.

This work continues previous research about multiple scattering of polarized light propagation in turbid media, putting emphasis on the imaginary part of the scatterers' complex refractive index. The whole angle-dependent Müller matrix is evaluated by comparing results of a polarization sensitive radiative transfer solution to Maxwell theory. Turbid media of defined scatterer concentrations are modelled in three dimensions by sphere ensembles kept inside a cubic or spherical simulation volume. This study addresses the impact of absorption on polarization characteristics for selected media from low to high absorption. Besides that, effects caused by multiple and dependent scattering are shown for increasing volume concentration. In this context some unique properties associated with multiple scattering and absorption are pointed out. Further, scattering results in two dimensions are compared for examples of infinite parallel cylinders of high absorption and perpendicularly incident plane waves.

Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels.

AIMS: The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. METHODS AND RESULTS: Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. CONCLUSIONS: A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics.

Fully automated spatially resolved reflectance spectrometer for the determination of the absorption and scattering in turbid media.

We describe a fully automated setup which is based on measurements of the spatially resolved reflectance for the determination of the reduced scattering and absorption coefficients in semi-infinite turbid media. The sample is illuminated with a xenon light source in combination with a monochromator enabling the scan of the wavelength from 450 nm to 950 nm. Reflected light from the sample is detected with a CCD camera providing a high spatial resolution. The essential steps for signal processing including, e.g., the consideration of the optical transfer function and the correct treatment of the background subtraction, are presented. The solutions of the diffusion theory and of the radiative transfer theory are investigated regarding the exact detection and illumination geometry. Systematic errors caused by using the different theories for fitting the optical parameters are characterized. The system was validated using liquid phantoms which contain Intralipid 20% and ink, and the measurement range of the system is specified. Further, we carefully characterized the optical properties of Intralipid 20% in the wavelength range between 450 nm and 950 nm.

Time-resolved optical spectroscopy of wood.

We have proposed and experimentally demonstrated that picosecond time-resolved optical spectroscopy in the visible/near-infrared (NIR) region (700-1040 nm) is a useful technique for noninvasive characterization of wood. This technique has been demonstrated on both softwood and hardwood samples treated in different ways simulating the aging process suffered by waterlogged woods. In all the cases, alterations of absorption and scattering spectra were observed, revealing changes of chemical and structural composition.

Magnification--a new look at a long-known optical property of dentin.

Light propagation in human dentin exhibits a strong directional dependence featuring the long-known optical magnification property. We hypothesized that this anisotropic effect is caused by multiple scattering at the dentin tubules, and not by fiberoptic effects, as had been previously assumed. We performed measurements of the transmitted intensity from dentin disks and compared them with Monte Carlo simulations of light propagation in dentin, considering the scattering by the tissue's microstructure. We found that the optical anisotropy of dentin can be fully explained with this model. We concluded that the magnification property of dentin is due to multiple scattering by the dental microstructure.

Anisotropy of light propagation in biological tissue.

We investigated the propagation of light in biological tissues that have aligned cylindrical microstructures (e.g., muscle, skin, bone, tooth). Because of pronounced anisotropic light scattering by cylindrical structures (e.g., myofibrils and collagen fibers) the spatially resolved reflectance exhibits a directional dependence that is different close to and far from the incident source. We applied Monte Carlo simulations, using the phase function of an infinitely long cylinder, to explain quantitatively the experimental results. These observations have consequences for noninvasive determination of the optical properties of tissue as well as for the diagnosis of early tissue alterations.

Treatment of idiopathic trigeminal neuralgia: comparison of long-term outcome after radiofrequency rhizotomy and microvascular decompression.

OBJECTIVE: To evaluate the long-term outcome of patients after either percutaneous trigeminal rhizotomy or microvascular decompression (MVD) for idiopathic trigeminal neuralgia at a single institution. METHODS: From 1977 to 1997, 316 radiofrequency lesion procedures and 378 MVDs were performed. Questionnaires were sent to all patients who were alive in 1981, 1982, 1992, and 1998. For all other patients, interviews were conducted with their relatives and general practitioners. A retrospective comparative analysis was performed with Kaplan-Meier probability curves as of the latest follow-up date. In addition, 80 patients who underwent MVD were examined postoperatively with quantitative sensory measurements by use of von Frey hairs. RESULTS: Two hundred twenty-five patients who underwent MVD and 206 patients who underwent radiofrequency could be analyzed retrospectively in detail. Overall, there was a 50% risk for recurrence of pain 2 years after percutaneous radiofrequency rhizotomy. Conversely, 64% of patients who underwent MVD remained completely pain free 20 years postoperatively. Patients without sensory impairment after MVD were pain free significantly longer than patients who experienced postoperative hypesthesia or partial rhizotomy. CONCLUSION: Because it is curative and nondestructive, MVD is considered the treatment of choice for trigeminal neuralgia in otherwise healthy people. In our study, it proved to be a more effective and long-lasting procedure for patients with typical trigeminal neuralgia than radiofrequency rhizotomy. Patients without postoperative sensory deficit remained pain free significantly longer, which is a strong argument against the "trauma" hypothesis of this procedure.

Non-invasive determination of muscle blood flow in the extremities from laser Doppler spectra.

We investigate theoretically the non-invasive determination of blood flow in muscles of the extremities using laser Doppler measurements. Laser Doppler spectra are calculated using Monte Carlo simulations and solutions of the correlation diffusion equation. The extremities are modelled as a two-layered turbid medium. The first layer represents the skin and subcutaneous fat layer and the second layer the muscle. It is shown that the absolute root-mean-square velocity of the blood in the muscle layer can be accurately derived in many practical cases if the laser Doppler spectra are measured at a distance which is sufficiently far from the source, and if the optical properties of the muscle are simultaneously determined.

Influence of the phase function on determination of the optical properties of biological tissue by spatially resolved reflectance.

Spatially resolved reflectance measurements are widely used for determination of the optical properties of biological media. However, the influence of the phase function on these measurements has not been quantified. We show that errors in the derived reduced scattering and absorption coefficients are as great as 100% for both absolute and relative spatially resolved reflectance measurements if a standard solution of the diffusion equation is used in the analysis. In addition, we investigated nonlinear regressions, using Monte Carlo simulations and an additional fitting parameter that characterizes the phase function, and found that the errors in the obtained optical coefficients were

Analysis of ultrashort feedback regulation in human placenta: synthesis and secretion of GnRH by human trophoblastic cells.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. AIM: Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. METHOD: First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. RESULTS: a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. CONCLUSIONS: GnRH is synthesised and released by trophoblastic cells.

In vivo determination of the optical properties of muscle with time-resolved reflectance using a layered model.

We have investigated the possibility of determining the optical coefficients of muscle in the extremities with in vivo time-resolved reflectance measurements using a layered model. A solution of the diffusion equation for two layers was fitted to three-layered Monte Carlo calculations simulating the skin, the subcutaneous fat and the muscle. Relative time-resolved reflectance data at two distances were used to derive the optical coefficients of the layers. We found for skin and subcutaneous fat layer thicknesses (l2) of up to 10 mm that the estimated absorption coefficients of the second layer of the diffusion model have differences of less than 20% compared with those of the muscle layer of the Monte Carlo simulations if the thickness of the first layer of the diffusion model is also fitted. If l2 is known, the differences are less than 5%, whereas the use of a semi-infinite model delivers differences of up to 55%. Even if l2 is only approximately known the absorption coefficient of the muscle can be determined accurately. Experimentally, the time-resolved reflectance was measured on the forearms of volunteers at two distances from the incident beam by means of a streak camera. The thicknesses of the tissues involved were determined by ultrasound. The optical coefficients were derived from these measurements by applying the two-layered diffusion model, and results in accordance with the theoretical studies were observed.

Noninvasive determination of the optical properties of two-layered turbid media.

Light propagation in two-layered turbid media having an infinitely thick second layer is investigated in the steady-state, frequency, and time domains. A solution of the diffusion approximation to the transport equation is derived by employing the extrapolated boundary condition. We compare the reflectance calculated from this solution with that computed with Monte Carlo simulations and show good agreement. To investigate if it is possible to determine the optical coefficients of the two layers and the thickness of the first layer, the solution of the diffusion equation is fitted to reflectance data obtained from both the diffusion equation and the Monte Carlo simulations. Although it is found that it is, in principle, possible to derive the optical coefficients of the two layers and the thickness of the first layer, we concentrate on the determination of the optical coefficients, knowing the thickness of the first layer. In the frequency domain, for example, it is shown that it is sufficient to make relative measurements of the phase and the steady-state reflectance at three distances from the illumination point to obtain useful estimates of the optical coefficients. Measurements of the absolute steady-state spatially resolved reflectance performed on two-layered solid phantoms confirm the theoretical results.

Investigation of two-layered turbid media with time-resolved reflectance.

Light propagation in two-layered turbid media that have an infinitely thick second layer is investigated with time-resolved reflectance. We used a solution of the diffusion equation for this geometry to show that it is possible to derive the absorption and the reduced scattering coefficients of both layers if the relative reflectance is measured in the time domain at two distances and if the thickness of the first layer is known. Solutions of the diffusion equation for semi-infinite and homogeneous turbid media are also applied to fit the reflectance from the two-layered turbid media in the time and the frequency domains. It is found that the absorption coefficient of the second layer can be more precisely derived for matched than for mismatched boundary conditions. In the frequency domain, its determination is further improved if phase and modulation data are used instead of phase and steady-state reflectance data. Measurements of the time-resolved reflectance were performed on solid two-layered tissue phantoms that confirmed the theoretical results.

Determination of the optical properties of semi-infinite turbid media from frequency-domain reflectance close to the source.

We investigate theoretically the errors in determining the reduced scattering and absorption coefficients of semi-infinite turbid media from frequency-domain reflectance measurements made at small distances between the source and the detector(s). The errors are due to the uncertainties in the measurement of the phase, the modulation and the steady-state reflectance as well as to the diffusion approximation which is used as a theoretical model to describe light propagation in tissue. Configurations using one and two detectors are examined for the measurement of the phase and the modulation and for the measurement of the phase and the steady-state reflectance. Three solutions of the diffusion equation are investigated. We show that measurements of the phase and the steady-state reflectance at two different distances are best suited for the determination of the optical properties close to the source. For this arrangement the errors in the absorption coefficient due to typical uncertainties in the measurement are greater than those resulting from the application of the diffusion approximation at a modulation frequency of 200 MHz. A Monte Carlo approach is also examined; this avoids the errors due to the diffusion approximation.

Improved solutions of the steady-state and the time-resolved diffusion equations for reflectance from a semi-infinite turbid medium.

Improved solutions of the diffusion equation for time-resolved and steady-state spatially resolved reflectance are investigated for the determination of the optical coefficients of semi-infinite turbid media such as tissue. These solutions are derived for different boundary conditions at the turbid-medium-air interface and are compared with Monte Carlo simulations. Relative reflectance data are fitted in the time domain, whereas relative and absolute reflectance are investigated in the steady-state domain. It is shown that the error in deriving the optical coefficients is, especially for steady-state spatially resolved reflectance, considerably smaller for the solutions under study than for the commonly used solutions. Analysis of experimental measurements of absolute steady-state spatially resolved reflectance confirms these results.