Wavelength dependence of crosstalk in dual-wavelength measurement of oxy- and deoxy-hemoglobin

In near-IR spectroscopy, the concentration change in oxy- and deoxyhemoglobin in tissue is calculated from the change in the detected intensity of light at two wavelengths by solving the simultaneous equation based on the modified Lambert-Beer law. The wavelength-independent constant or mean optical path length is usually assigned to the term of partial optical path length in the simultaneous equation. This insufficient optical path length in the calculation causes crosstalk between the concentration change in oxy- and deoxyhemoglobin. We investigate the crosstalk in the dual-wavelength measurement of oxy- and deoxyhemoglobin theoretically by Monte Carlo simulation to discuss the optimal wavelength pair to minimize the crosstalk. The longer wavelength of the dual-wavelength measurement is fixed at 830 nm and the shorter wavelength is varied from 650 to 780 nm. The optimal wavelength range for pairing with 830 nm for the dual-wavelength measurement of oxy- and deoxyhemoglobin is from 690 to 750 nm. The mean optical path length, which can be obtained by time- and phase-resolved measurement, is effective to reduce the crosstalk in the results of dual-wavelength measurement.     

Insulin-like growth factor I partly prevents axon elimination in the neonate rat optic nerve

Low-power, near-infra-red laser irradiation has been used to relieve patients from various kinds of pain, though the precise mechanisms of such biological actions of the laser have not yet been resolved. To investigate the cellular mechanisms by near-infra-red laser on the nervous system, we examined the effect of 830-nm laser irradiation on the energy metabolism of the rat brain. The diode laser was applied for 15 min with an irradiance of 4.8 W/cm(2). Tissue adenosine triphosphate (ATP) content of the irradiated area in the cerebral cortex was 19% higher than that of the non-treated area, whereas the adenosine diphosphate (ADP) content showed no significant difference. Laser irradiation at another wavelength (652 nm) had no effect on either ATP or ADP contents. The temperature of the tissue was increased by 4.4-4.7 degrees C during the irradiation of both wavelengths. These results suggest that the increase in tissue ATP content did not result from the thermal effect, but from a specific effect of the laser operated at the 830-nm wavelength.     

Optical imaging as an adjunct to sonograph in differentiating benign from malignant breast lesions

The role of near infrared (NIR) diffusive light imaging as an adjunct to ultrasound in differentiating benign from malignant lesions was evaluated in 27 mammography patients with infiltrating ductal carcinomas, apocrine metaplasia, fibroadenomas, radial scar and ductal hyperplasia, cysts, and normal tissues. Conventional ultrasound/mammography images were graded based on BI-RADS assessment categories. The spatial NIR measurements were made at wavelengths of 750 and 830 nm. Functional images, such as relative changes of deoxyhemoglobin (deoxyHb) and total blood concentration, were estimated from the dual wavelength measurements. Maximum relative deoxyHb and blood concentration changes were measured, and spatial correlation of masses in relative deoxyHb and blood concentration images for each breast were calculated. For the five biopsy proven benign lesions, ultrasound/mammography diagnoses were suspicious for malignancy (four cases) and highly suspicious for malignancy (one case). Four lesions showed less than 1.0 V maximum deoxyHb and less than 1.5 V maximum blood concentration levels on average and spatial image correlation showed no correlated masses in both deoxyHb and blood concentration images. For the four biopsy proven malignant lesions, ultrasound/mammography diagnoses were highly suspicious for malignancy. Maximum deoxyHb and blood concentration changes were greater than 2.9 V on average except one lesion which showed smaller deoxyHb signal (maximum 0.85 V) but the deoxyHb mass and blood concentration mass were highly correlated.     

Linear birefringence of the retinal nerve fiber layer measured in vitro with a multispectral imaging micropolarimeter

Scanning laser polarimetry (SLP) assesses the retinal nerve fiber layer (RNFL) for glaucoma diagnosis by detecting the birefringence of the peripapillary RNFL. A detailed understanding of SLP requires an accurate value for RNFL birefringence in order to relate measured retardance to RNFL thickness, but current knowledge of this value is limited. A multispectral imaging micropolarimeter of PSC'A type was used to measure the retardance in transmission of the RNFL of isolated rat retina before (living) and after (fixed) 20 min of glutaraldehyde fixation. The thickness of the nerve fiber bundles measured was then determined histologically. As previously known from reflectance measurements, in transmission the RNFL behaved as a linear retarder. The retardance of the RNFL was constant at wavelengths from 440 to 830 nm and persisted after tissue fixation. In 37 nerve fiber bundles of 8 retinas, the average RNFL birefringence was 0.23 nm/microm before and 0.19 nm/microm after fixation, with an uncertainty of 0.01 nm/microm. The wavelength independence is consistent with a mechanism of form birefringence from thin cylindrical organelles. These results allow extrapolation of previous visible wavelength measurements to the near-infrared wavelengths used by SLP and validate the use of fixed tissue for RNFL research.     

Resonance Raman spectroscopy of optically trapped functional erythrocytes

We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.     

Limitation on sensitivity of measuring collagen degradation: studies with cultured liver cells

Intracellular degradation of newly synthesized collagen is studied by incubating cells with (14C)proline and measuring the hydroxy(14C)proline in a low molecular weight fraction relative to total hydroxy-(14C)proline synthesized. This communication describes a fundamental limitation on the precision with which the measurement can be made; the limitation derives from an irreducible uncertainty in estimating the background radioactivity contributed by the isotope used for metabolic labeling. The problem is illustrated by experiments with cultured liver cells. In rat hepatocytes, the hydroxy-(14C)proline in the low molecular weight fraction was barely detectable above background; the degradation was 29%, however the uncertainty was +/- 28%. In contrast, significant amounts of hydroxy(14C)proline were detected in fractions from human and rat hepatoma cells; and while the levels of degradation were approximately the same as in rat hepatocytes, the measurements could be made with substantially greater precision.     

Objective Interpretation of the Rapid Urease Test for Helicobacter pylori Infection Using Colorimetry

BackgroundThe rapid urease test (RUT) is a major diagnostic tool for detecting Helicobacter pylori infection. This study aimed to establish an objective method for measuring the color changes in the RUT kit to improve the test’s diagnostic accuracy.MethodsA UV-visible spectrophotometer was selected as the colorimeter; experiments were conducted in three stages to objectively identify the color changes in the RUT kit.ResultsFirst, the urea broth solution showed an identifiable color change from yellow to red as the pH increased by 0.2. The largest transmittance difference detected using the UV-visible spectrophotometer was observed at a 590-nm wavelength. Second, the commercialized RUT kit also showed a gradual color change according to the pH change detected using the UV-visible spectrophotometer. Third, 13 cases of negative RUT results with a biopsy specimen and 16 of positive RUT results were collected. The transmittance detected using the UV-visible spectrophotometer showed a clear division between the positive and negative RUT groups; the largest difference was observed at a 559-nm wavelength. The lowest transmittance in the negative RUT group was 64, while the highest in the positive RUT group was 56, at the 559-nm wavelength. The UV-visible spectrophotometry reading showed a consistency of 92.7% compared with that of manual reading.ConclusionA transmittance of 60 at a 559-nm wavelength detected using UV-visible spectrophotometer can be used as a cutoff value for interpreting RUT results; this will help develop an automatic RUT kit reader with a high accuracy.     

L-shell x-ray fluorescence measurements of lead in bone: accuracy and precision

This study aimed to quantify the accuracy and precision of a method for in vivo measurements of lead in bone using L-shell x-ray fluorescence (LXRF), the former via comparison with independent measurements of lead in bone obtained using electrothermal atomic absorption spectrometry (AAS) following acid digestion. Using LXRF. the lead content of adult human cadaver tibiae was measured, both as intact legs and as dissected tibiae with overlying tissue removed, the latter at several proximal-distal locations. After LXRF, each tibia was divided into nine cross-sectional segments, which were further separated into tibia core and surface samples for AAS measurement. The proximal-distal variability of AAS-measured core and surface tibia lead concentrations has been described elsewhere (the lead concentration was found to decrease towards both ends of the tibia). The subjects of this paper are the proximal-distal variability of the LXRF-measured lead concentrations, the measurement uncertainty and the statistical agreement between LXRF and AAS. There was no clear proximal-distal variability in the LXRF-measured concentrations; the degree of variability in actual tibia lead concentrations is far less than the LXRF measurement uncertainty. Measurement uncertainty was dominated by counting statistics and exceeded the estimate of lead concentration in most cases. The agreement between LXRF and AAS was reasonably good for bare bone measurements but poor for intact leg measurements. The variability of the LXRF measurements was large enough, for both bare bone and intact leg measurements, to yield grave concerns about the analytical use of the technique in vivo.     

Wavelength optimization for rapid chromophore mapping using spatial frequency domain imaging

Spatial frequency-domain imaging (SFDI) utilizes multiple-frequency structured illumination and model-based computation to generate two-dimensional maps of tissue absorption and scattering properties. SFDI absorption data are measured at multiple wavelengths and used to fit for the tissue concentration of intrinsic chromophores in each pixel. This is done with a priori knowledge of the basis spectra of common tissue chromophores, such as oxyhemoglobin (ctO(2)Hb), deoxyhemoglobin (ctHHb), water (ctH(2)O), and bulk lipid. The quality of in vivo SFDI fits for the hemoglobin parameters ctO(2)Hb and ctHHb is dependent on wavelength selection, fitting parameters, and acquisition rate. The latter is critical because SFDI acquisition time is up to six times longer than planar two-wavelength multispectral imaging due to projection of multiple-frequency spatial patterns. Thus, motion artifact during in vivo measurements compromises the quality of the reconstruction. Optimal wavelength selection is examined through matrix decomposition of basis spectra, simulation of data, and dynamic in vivo measurements of a human forearm during cuff occlusion. Fitting parameters that minimize cross-talk from additional tissue chromophores, such as water and lipid, are determined. On the basis of this work, a wavelength pair of 670 nm∕850 nm is determined to be the optimal two-wavelength combination for in vivo hemodynamic tissue measurements provided that assumptions for water and lipid fractions are made in the fitting process. In our SFDI case study, wavelength optimization reduces acquisition time over 30-fold to 1.5s compared to 50s for a full 34-wavelength acquisition. The wavelength optimization enables dynamic imaging of arterial occlusions with improved spatial resolution due to reduction of motion artifacts.     

Two-wavelength carbon dioxide laser application for in-vitro blood glucose measurements

To develop a fast and easy clinical method for glucose measurements on whole blood samples, changes in glucose spectra are investigated varying temperature, glucose concentration, and solvent using attenuated total reflection Fourier transform infrared (ATR- FTIR) measurements. The results show a stability of the spectra at different temperatures and wavelength shifts of the absorption bands when water is replaced by blood. Because the ATR measurements are influenced by sedimentation of the red blood cells, a two-wavelength CO2 laser is used to determine the glucose concentration in whole blood samples. For this purpose, the first laser wavelength lambda(1) is tuned to the maximum of the glucose absorption band in blood at 1080 cm(-1), and the second laser wavelength lambda 2 is tuned to 950 cm(-1) for background measurements. The transmitted laser power through the optical cell containing the whole blood sample at lambda 1 and lambda 2 is used to determine the ratio. This signal correlates well with the glucose concentration in the whole blood samples. The CO2 laser measurement is too fast to be influenced by the red blood cell sedimentation, and will be a suitable method for glucose determination in whole blood.     

Evaluation of accuracy and precision of adenovirus absorptivity at 260 nm under conditions of complete DNA disruption

A simple, accurate, and precise method to determine adenovirus particle concentration using 260-nm absorbance was developed as an enhancement to the method of Maizel et al. (1968, Virology 36, 115-125). This modified method ensures complete disruption of virus particles and viral DNA prior to absorbance measurements, therefore eliminating absorbance measurement errors due to hyperchromic shift and thus providing an extinction coefficient at 260 nm that is directly related to protein concentration as measured by the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275). Application of this modified method will reduce interlaboratory variability in determining adenovirus particle concentrations, as current practices reflected in the literature utilize varying sample preparation procedures and calculation methods which cause underestimation of adenovirus concentration by almost twofold. The sample pretreatment conditions used in this modified method entail incubation in 1% sodium dodecyl sulfate at 100 degrees C for 4 min and result in an adenovirus 260-nm absorptivity of 1.8 x 10(12) viral particles per milliliter per absorbance unit in a 1-cm-pathlength cell.     

Precise radiochromic film dosimetry using a flat-bed document scanner

In this study, a measurement protocol is presented that improves the precision of dose measurements using a flat-bed document scanner in conjunction with two new GafChromic film models, HS and Prototype A EBT exposed to 6 MV photon beams. We established two sources of uncertainties in dose measurements, governed by measurement and calibration curve fit parameters contributions. We have quantitatively assessed the influence of different steps in the protocol on the overall dose measurement uncertainty. Applying the protocol described in this paper on the Agfa Arcus II flat-bed document scanner, the overall one-sigma dose measurement uncertainty for an uniform field amounts to 2% or less for doses above around 0.4 Gy in the case of the EBT (Prototype A), and for doses above 5 Gy in the case of the HS model GafChromic film using a region of interest 2 X 2 mm2 in size.     

The Spectralab-M quadrupole medical mass spectrometer

A practical universal gas concentration measuring instrument would have great advantages as the basis of many physiological measurements. The mass spectrometer offers such an instrument but difficulty in calibration has made it inaccessible to all but the most technically minded department. This paper reports measurements on the Spectralab-M, a medical mass spectrometer that offers easy use and calibration by using microprocessor control. The performance of the machine configured for respiratory measurements gave an accuracy better than 0.03 +/- 0.1% vol/vol. Configured for anaesthetic gas measurements the instrument gave an accuracy better than 0.07 +/- 0.36% vol/vol for all gases. In both cases no systematic alinearities were observed. The speed of response of the machine was measured with a standard heated stainless-steel capillary inlet of 1.5 m. Response times (10-90%) of less than 100 ms were obtained on all gases measured with typical lag times of 500 ms at a flow rate of 15 ml min-1.     

Fluorescence properties of albumin blue 633 and 670 in plasma and whole blood

We have determined the fluorescence characteristics of two long wavelength dyes, albumin blue 633 (AB633) and 670 (AB670), in plasma and blood to evaluate the possibility of making direct fluorescence sensing measurements in blood. Using binding and lifetime measurements we were also able to show that these dyes bind selectively to human serum albumin (HSA) in plasma and blood. By measuring changes in the mean lifetime of AB670 with changes in the HSA concentration, we showed that lifetime-based sensing can be used to monitor HSA concentrations using these albumin blue dyes. Anisotropy measurements for AB633 and AB670 in plasma and blood revealed high anisotropy values for these dyes in these media. Exploiting these high anisotropies, we were also able to determine HSA concentrations in plasma and blood mimics using changes in AB670 anisotropy with HSA concentration. These results show that, apart from being able to make fluorescence measurements directly in plasma and blood, it is possible to sense directly for specific plasma/blood components using fluorescent probes that bind preferentially to them.     

Determination of lamotrigine in human plasma by high-performance liquid chromatography.

The method involves precipitation of plasma proteins with acetonitrile and analysis of the supernatant by high-performance liquid chromatography using a 5 microm Zorbax C8 column. Quantitation was performed by measurement of the UV absorbance at a wavelength of 306 nm. The method was linear in the range of 1-20 microg/ml, with a mean coefficient of determination (r2=0.998). The limit of detection was 0.6 microg/ml and the lower limit of quantitation was 1 microg/ml using 200 microl of plasma. Within- and between-day accuracy and precision were below 6% at all analysed concentrations except at the limit of quantitation. No interfering peaks were found by commonly monitored antiepileptic drugs. Recovery was found to be > or =99%. Satisfactory performance was obtained in the evaluation of epileptic patient samples, whose results of plasma concentration measurements are briefly discussed. We conclude that this is a reliable method for the routine monitoring of lamotrigine concentration in plasma in the clinical setting.     

Single spin-echo proton transverse relaxometry of iron-loaded liver

A single-spin-echo methodology is described for the measurement and imaging of proton transverse relaxation rates (R2) in iron-loaded and normal human liver tissue in vivo. The methodology brings together previously reported techniques dealing with (i) the changes in gain between each spin-echo acquisition, (ii) signal level offset due to background noise, (iii) estimation of signal intensities in decay curves at time zero to enable reliable extraction of relaxation times from tissues with very short T2 values, (iv) bi-exponential modelling of decay curves with a small number of data points, and (v) reduction of respiratory motion artefacts. The accuracy of the technique is tested on aqueous manganese chloride solutions yielding a relaxivity of 74.1+/-0.3 s-1 (mM)-1, consistent with previous reports. The precision of the in vivo measurement of mean liver R2 values is tested through duplicate measurements on 10 human subjects with mean liver R2 values ranging from 26 to 220 s-1. The random uncertainty on the measurement of mean liver R2 was found to be 7.7%.     

Measurement of lung density by photon transmission for monitoring intravascular and extravascular fluid volume changes in the lungs.

Left ventricular (LV) heart failure increases pulmonary blood volume (PBV) and interstitial fluid volume. Continuous measurements of lung density may be a simple non-invasive method for monitoring of the LV function as the lung density should reflect the changes in the PBV and extravascular lung water. The purpose of the study was to optimize transmission measurements of the lung with a gamma camera and a planar source. Another aim was to assess the possibility of transmission monitoring in normal subjects at rest and during exercise and to compare the results with simultaneous measurements of PBV changes. Transmission measurements were made in a water-filled phantom containing lungs of different density. A gamma camera and a planar 57Co source were used. The coefficient of variation in density determination owing to counting statistics in a lung area was calculated for different energy windows, acquisition times and collimation. Dynamic measurements in normal subjects were carried out in a sitting and a supine position at rest and during exercise. Pulmonary blood volume was monitored simultaneously using 99mTc-labelled red blood cells and the registered blood pool activity was corrected for attenuation. Highest precision in relative density determination was obtained with large energy window and uncollimated source. A precision of 1.0% was obtained with 1 min measuring time. About 10% change in transmission corresponding to a 15% change in density was observed during exercise. Changes in blood pool and lung density covariated. We conclude that lung density changes can be monitored with a high degree of statistical precision in a few minutes and with a low radiation dose of radiation using a gamma camera and a planar source.     

The radial depth-dose distribution of a 188W/188Re beta line source measured with novel,ultra-thin TLDs in a PMMA phantom: comparison with Monte Carlo simulations

The radial depth-dose distribution of a prototype 188W/188Re beta particle line source of known activity has been measured in a PMMA phantom, using a novel, ultra-thin type of LiF:Mg,Cu,P thermoluminescent detector (TLD). The measured radial dose function of this intravascular brachytherapy source agrees well with MCNP4C Monte Carlo simulations, which indicate that 188Re accounts for > or = 99% of the dose between 1 mm and 5 mm radial distance from the source axis. The TLDs were calibrated using a 90Sr/90Y beta secondary standard. Several correction factors are calculated using analytical and Monte Carlo methods. An analysis of the measurement uncertainty is made. Since it is partly determined by components of uncertainty arising from random effects, repeated measurements yield a lower uncertainty. The expanded uncertainty in the absolute dose at 2 mm radial distance equals 11%, 10%, 9% and 8% for 1, 2, 3 and 5 measurements, respectively. After a correction for source non-uniformity, the measured dose rate per unit source activity at 2 mm radial distance equals (1.53 +/- 0.16) Gy min(-1) GBq(-1) (2sigma), in agreement with the value of (1.45 +/- 0.01) Gy min(-1) GBq(-1) (2sigma) predicted by the MCNP4C simulations.     

Design and implementation of a multifrequency near-infrared diffuse optical tomography system

The design and implementation of a multifrequency and multispectral diffuse optical tomography system is described. Four wavelengths are utilized: 665, 785, 808, and 830 nm. The system is based on a network analyzer, which provides rf modulation signals for the laser diodes, as well as measures the amplitude and the phase of the detected signals. Six different modulation frequencies ranging from 110 to 280 MHz are used. The details of instrumentation, calibration, data acquisition, and performance of the system are given. A finite element algorithm is used to solve the diffusion equation, and an inverse solver based on this forward solver is implemented to calculate the absorption and scattering maps from the acquired data. Data acquisition for one wavelength is completed in less than 2.5 min for a single modulation frequency. The measurement repeatability is 0.5% in ac intensity and 0.2 deg in phase. The performance of the system is evaluated with phantom studies. A multifrequency reconstruction algorithm is used, in which a single absorption and scattering image pair is obtained using the whole dataset obtained at different modulation frequencies. It is shown that the multifrequency reconstruction approach provides superior image quality compared to the single frequency counterpart.