Comparison of spatially and temporally resolved diffuse-reflectance measurement systems for determination of biomedical optical properties

Time-resolved and spatially resolved measurements of the diffuse reflectance from biological tissue are two well-established techniques for extracting the reduced scattering and absorption coefficients. We have performed a comparison study of the performance of a spatially resolved and a time-resolved instrument at wavelengths 660 and 786 nm and also of an integrating-sphere setup at 550-800 nm. The first system records the diffuse reflectance from a diode laser by means of a fiber bundle probe in contact with the sample. The time-resolved system utilizes picosecond laser pulses and a single-photon-counting detection scheme. We extracted the optical properties by calibration using known standards for the spatially resolved system, by fitting to the diffusion equation for the time-resolved system, and by using an inverse Monte Carlo model for the integrating sphere. The measurements were performed on a set of solid epoxy tissue phantoms. The results showed less than 10% difference in the evaluation of the reduced scattering coefficient among the systems for the phantoms in the range 9-20 cm(-1), and absolute differences of less than 0.05 cm(-1) for the absorption coefficient in the interval 0.05-0.30 cm(-1).     

Monte Carlo-based inverse model for calculating tissue optical properties. Part I: Theory and validation on synthetic phantoms

A flexible and fast Monte Carlo-based model of diffuse reflectance has been developed for the extraction of the absorption and scattering properties of turbid media, such as human tissues. This method is valid for a wide range of optical properties and is easily adaptable to existing probe geometries, provided a single phantom calibration measurement is made. A condensed Monte Carlo method was used to speed up the forward simulations. This model was validated by use of two sets of liquid-tissue phantoms containing Nigrosin or hemoglobin as absorbers and polystyrene spheres as scatterers. The phantoms had a wide range of absorption (0-20 cm(-1)) and reduced scattering coefficients (7-33 cm(-1)). Mie theory and a spectrophotometer were used to determine the absorption and reduced scattering coefficients of the phantoms. The diffuse reflectance spectra of the phantoms were measured over a wavelength range of 350-850 nm. It was found that optical properties could be extracted from the experimentally measured diffuse reflectance spectra with an average error of 3% or less for phantoms containing hemoglobin and 12% or less for phantoms containing Nigrosin.     

Spatially resolved absolute diffuse reflectance measurements for noninvasive determination of the optical scattering and absorption coefficients of biological tissue

The absorption and transport scattering coefficients of biological tissues determine the radial dependence of the diffuse reflectance that is due to a point source. A system is described for making remote measurements of spatially resolved absolute diffuse reflectance and hence noninvasive, noncontact estimates of the tissue optical properties. The system incorporated a laser source and a CCD camera. Deflection of the incident beam into the camera allowed characterization of the source for absolute reflectance measurements. It is shown that an often used solution of the diffusion equation cannot be applied for these measurements. Instead, a neural network, trained on the results of Monte Carlo simulations, was used to estimate the absorption and scattering coefficients from the reflectance data. Tests on tissue-simulating phantoms with transport scattering coefficients between 0.5 and 2.0 mm(-1) and absorption coefficients between 0.002 and 0.1 mm(-1) showed the rms errors of this technique to be 2.6% for the transport scattering coefficient and 14% for the absorption coefficients. The optical properties of bovine muscle, adipose, and liver tissue, as well as chicken muscle (breast), were also measured ex vivo at 633 and 751 nm. For muscle tissue it was found that the Monte Carlo simulation did not agree with experimental measurements of reflectance at distances less than 2 mm from the incident beam.     

In vivo local determination of tissue optical properties: applications to human brain

Local and superficial near-infrared (NIR) optical-property characterization of turbid biological tissues can be achieved by measurement of spatially resolved diffuse reflectance at small source-detector separations (<1.4 mm). However, in these conditions the inverse problem, i.e., calculation of localized absorption and the reduced scattering coefficients, is necessarily sensitive to the scattering phase function. This effect can be minimized if a new parameter of the phase function gamma, which depends on the first and the second moments of the phase function, is known. If gamma is unknown, an estimation of this parameter can be obtained by the measurement, but the uncertainty of the absorption coefficient is increased. A spatially resolved reflectance probe employing multiple detector fibers (0.3-1.4 mm from the source) is described. Monte Carlo simulations are used to determine gamma, the reduced scattering and absorption coefficients from reflectance data. Probe performance is assessed by measurements on phantoms, the optical properties of which were measured by other techniques [frequency domain photon migration (FDPM) and spatially resolved transmittance]. Our results show that changes in the absorption coefficient, the reduced scattering coefficient, and gamma can be measured to within +/-0.005 mm(-1), +/-0.05 mm(-1), and +/-0.2, respectively. In vivo measurements performed intraoperatively on a human skull and brain are reported for four NIR wavelengths (674, 811, 849, 956 nm) when the spatially resolved probe and FDPM are used. The spatially resolved probe shows optimum measurement sensitivity in the measurement volume immediately beneath the probe (typically 1 mm(3) in tissues), whereas FDPM typically samples larger regions of tissues. Optical-property values for human skull, white matter, scar tissue, optic nerve, and tumors are reported that show distinct absorption and scattering differences between structures and a dependence on the phase-function parameter gamma.     

Determination of the optical coefficients of biological tissue by neural network

A system that incorporated a laser source and a CCD camera was used to measure spatially-resolved steady-state diffuse reflectance. Monte Carlo simulations and experiments in tissue phantoms were used to train a neural network that characterizes the reflectance data on a turbid medium. The neural network was used to extract the optical properties (scattering and absorption coefficients) of biological tissue. The accuracy of the neural network was investigated and validated. Tests on tissue-simulation phantoms showed the relative errors of this technique to be 3% for the reduced scattering coefficient and 9% for the absorption coefficients. The optical properties of human skin were also measured in vivo at 633 nm. For human skin tissue it was found that our results were in good agreement with their reference values.     

Experimental and theoretical evaluation of a fiber-optic approach for optical property measurement in layered epithelial tissue

Improvements in measurement of epithelial tissue optical properties (OPs) in the ultraviolet and visible (UV-Vis) may lead to enhanced understanding of optical techniques for neoplasia detection. In this study, we investigated an approach based on fiber-optic measurement of reflectance to determine absorption and reduced scattering coefficients (μ(a) and μ(s)') in two-layer turbid media. Neural network inverse models were trained on simulation data for a wide variety of OP combinations (μ(a) = 1-22.5, μ(s)' = 5-42.5 cm(-1)). Experimental measurements of phantoms with top-layer thicknesses (D) ranging from 0.22 to 0.66 mm were performed at three UV-Vis wavelengths. OP estimation accuracy was calculated and compared to theoretical results. Mean prediction errors were strongly correlated with D and ranged widely, from 1.5 to 12.1 cm(-1). Theoretical analyses indicated the potential for improving accuracy with alternate probe geometries. Although numerous challenges remain, this initial experimental study of an unconstrained approach for fiber-optic-based OP determination in two-layer epithelial tissue indicates the potential to provide useful measurements.     

Hyperspectral diffuse reflectance imaging for rapid, noncontact measurement of the optical properties of turbid materials

We present a method and technique of using hyperspectral diffuse reflectance for rapid determination of the optical properties of turbid media. A hyperspectral imaging system in line scanning mode was used to acquire spatial diffuse reflectance profiles from liquid phantoms made up of absorbing dyes and fat emulsion scatterers over the spectral range of 450-1000 nm instantaneously. The hyperspectral reflectance data were analyzed by using a steady-state diffusion approximation model for semi-infinite homogeneous media. A calibration procedure was developed to compensate the nonuniform instrument response of the imaging system, and a curve-fitting algorithm was used to extract absorption and reduced scattering coefficients (mua and mus', respectively) for the phantoms in the wavelength range from 530 to 900 nm. The hyperspectral imaging system gave good measures of mua and mus' for the phantoms with average fitting errors of 12% and 7%, respectively. The hyperspectral imaging technique is fast, noncontact, and easy to use, which makes it especially suitable for measurement of the optical properties of turbid liquid and solid foods.     

Optical properties of normal and carcinomatous bronchial tissue

To understand better the optical characteristics and autofluorescence properties of normal and carcinomatous bronchial tissue, we measured the absorption coefficient, scattering coefficient, and anisotropy factor from 400 to 700 nm. We made the measurements by using an integrating sphere with a collimated white-light beam to measure total reflectance and transmittance of samples. The unscattered transmittance of the samples was measured through polarized on-axis light detection. The inverse adding-doubling solution was utilized to solve the equation of radiative transfer and to determine the absorption coefficient and reduced scattering coefficient. The scattering coefficient and anisotropy factor were derived from the unscattered transmittance of the sample and the reduced scattering coefficient. The measured parameters allow us to simulate photon propagation in normal bronchial and tumoral tissue by using Monte Carlo modeling.     

Determination of optical coefficients of biological tissue from a single integrating-sphere

The detection of interactions between light and tissue can be used to characterize the optical properties of the tissue. The development is described of a method that determines optical coefficients of biological tissue from a single optical reflectance spectrum measured with an integrating-sphere. The experimental system incorporated a DH-2000 deuterium tungsten halogen light source, a USB4000-VIS-NIR miniature fiber optic spectrometer and an integrating-sphere. Fat emulsion and ink were used to mimic the scattering and absorbing properties of tissue in the tested sample. The measured optical reflectance spectrums with different scattering and absorbing properties were used to train a back-propagation neural network (BPNN). Then the neural network (BPNN) was used to determine the optical coefficients of biological tissue from a single optical reflectance spectrum measured with an integrating-sphere. Tests on tissue-simulation phantoms showed the relative errors of this technique to be 7% for the reduced scattering coefficient and 15% for the absorption coefficients. The optical properties of human skin were also measured in vivo.     

Measurement of the absorption and scattering properties of turbid liquid foods using hyperspectral imaging

A hyperspectral imaging system in line scanning mode was used for measuring the absorption and scattering properties of turbid food materials over the visible and near-infrared region of 530-900 nm. An instrumental calibration procedure was developed to compensate for the nonuniform instrument response of the imaging system. A nonlinear curve-fitting algorithm for a steady-state diffusion theory model was proposed to determine absorption (mua) and reduced scattering coefficients (mu's) from the spatially resolved hyperspectral reflectance profiles. The hyperspectral imaging system provided good measurement of mua and mu's for the simulation samples made of Intralipid scattering material and three absorbers (blue dye, green dye, and black ink) with average fitting errors of 16% and 11%, respectively. The optical properties of the fruit and vegetable juices and milks were determined. Values of the absorption and reduced scattering coefficient at 600 nm were highly correlated to the fat content of the milk samples with the correlation coefficient of 0.995 and 0.998, respectively. Compared to time-resolved and frequency-domain techniques, the hyperspectral imaging technique provides a faster and simpler means for measuring the optical properties of turbid food and agricultural products.     

Determination of optical parameters of human breast tissue from spatially resolved fluorescence: a diffusion theory model

We report the measurement of optical transport parameters of pathologically characterized malignant tissues, normal tissues, and different types of benign tumors of the human breast in the visible wavelength region. A spatially resolved steady-state diffuse fluorescence reflectance technique was used to estimate the values for the reduced-scattering coefficient (mu(s)') and the absorption coefficient (mu(a)) of human breast tissues at three wavelengths (530, 550, and 590 nm). Different breast tissues could be well differentiated from one another, and different benign tumors could also be distinguished by their measured transport parameters. A diffusion theory model was developed to describe fluorescence light energy distribution, especially its spatial variation in a turbid and multiply scattering medium such as human tissue. The validity of the model was checked with a Monte Carlo simulation and also with different tissue phantoms prepared with polystyrene microspheres as scatterers, riboflavin as fluorophores, and methylene blue as absorbers.     

Measurement of optical transport properties of normal and malignant human breast tissue

We report measurement of optical transport parameters of normal and malignant (ductal carcinoma) human breast tissue. A spatially resolved steady-state diffuse reflectance technique was used for measurement of the reduced scattering coefficient (mu(s)?) and the absorption coefficient (mu(a)) of the tissue. The anisotropy parameter of scattering (g) was estimated by goniophotometric measurements of the scattering phase function. The values of mu(s)? and mu(a) for malignant breast tissue were observed to be larger than those for normal breast tissue over the wavelength region investigated (450-650 nm). Further, by using both the diffuse reflectance and the goniophotometric measurements, we estimated the Mie equivalent average radius of tissue scatterers to be larger in malignant tissue than in normal tissue.     

Imaging with diffusing light: an experimental study of the effect of background optical properties

The overall image quality and diagnostic potential of time-resolved transmittance imaging depend on sensitivity to optical contrast, capacity to discriminate scattering from absorption contributions, and spatial resolution. We have investigated experimentally the effects of the optical properties of the background medium on the overall image quality of optical imaging based on fitting the experimental data to the solution of the diffusion equation and on time gating. Images were acquired from phantoms with different background optical properties, while the optical contrast between inhomogeneities and background is kept constant. Data were collected every 0.2 cm over a 6 cm x 6 cm area from realistic tissue phantoms containing cylindrical inhomogeneities (1 cm high and 1 cm in diameter) embedded in a 5-cm-thick turbid slab. The optical coefficients of the background were varied in the ranges of 5-15 cm(-1) for transport scattering and 0.02-0.08 cm(-1) for absorption. The optical contrast for the inclusions was kept at values of -50% and +50% for the scattering and -75% and +300% for the absorption. The results show that both high scattering and high absorption are beneficial.     

Real-time absorption and scattering characterization of slab-shaped turbid samples obtained by a combination of angular and spatially resolved measurements

We present a fast and accurate method for real-time determination of the absorption coefficient, the scattering coefficient, and the anisotropy factor of thin turbid samples by using simple continuous-wave noncoherent light sources. The three optical properties are extracted from recordings of angularly resolved transmittance in addition to spatially resolved diffuse reflectance and transmittance. The applied multivariate calibration and prediction techniques are based on multiple polynomial regression in combination with a Newton--Raphson algorithm. The numerical test results based on Monte Carlo simulations showed mean prediction errors of approximately 0.5% for all three optical properties within ranges typical for biological media. Preliminary experimental results are also presented yielding errors of approximately 5%. Thus the presented methods show a substantial potential for simultaneous absorption and scattering characterization of turbid media.     

Measurement of the optical properties of a two-layer model of the human head using broadband near-infrared spectroscopy

We present the development of a continuous-wave method of quantifying the optical properties of a two-layered model of the human head using a broadband spectral approach. Absolute absorption and scattering properties of the upper and lower layers of phantoms with known optical properties were reconstructed from steady-state multi-distance measurements by performing differential fit analysis of the near-infrared reflectance spectrum between 700 and 1000 nm. From spectra acquired at 10, 20, and 30 mm, the concentration of a chromophore in the bottom layer was determined within an error of 10% in the presence of a 15 mm thick top layer. These results demonstrate that our method was able to determine the optical properties of the lower layer, which represents brain, with acceptable error at specific source-detector distances.     

Fluorescence spectra provide information on the depth of fluorescent lesions in tissue

The fluorescence spectrum measured from a fluorophore in tissue is affected by the absorption and scattering properties of the tissue, as well as by the measurement geometry. We analyze this effect with Monte Carlo simulations and by measurements on phantoms. The spectral changes can be used to estimate the depth of a fluorescent lesion embedded in the tissue by measurement of the fluorescence signal in different wavelength bands. By taking the ratio between the signals at two wavelengths, we show that it is possible to determine the depth of the lesion. Simulations were performed and validated by measurements on a phantom in the wavelength range 815-930 nm. The depth of a fluorescing layer could be determined with 0.6-mm accuracy down to at least a depth of 10 mm. Monte Carlo simulations were also performed for different tissue types of various composition. The results indicate that depth estimation of a lesion should be possible with 2-3-mm accuracy, with no assumptions made about the optical properties, for a wide range of tissues.     

Analytical model of light reflectance for extraction of the optical properties in small volumes of turbid media

Monte Carlo simulations and experiments in tissue phantoms were used to empirically develop an analytical model that characterizes the reflectance spectrum in a turbid medium. The model extracts the optical properties (scattering and absorption coefficients) of the medium at small source-detector separations, for which the diffusion approximation is not valid. The accuracy of the model and the inversion algorithm were investigated and validated. Four fiber probe configurations were tested for which both the source and the detector fibers were tilted at a predetermined angle, with the fibers parallel to each other. This parallel-fiber geometry facilitates clinical endoscopic applications and ease of fabrication. Accurate extraction of tissue optical properties from in vivo spectral measurements could have potential applications in detecting, noninvasively and in real time, epithelial (pre)cancers.     

Features of the attenuation and single-sided imaging potential of near-infrared laser radiation in tissue-like liquid turbid media

Tissue-like phantoms are important tools in studying light propagation and scattering in biological tissues and in the development, testing and calibration of novel optical diagnostic and therapeutic methods and instruments. This motivates the interest in characterizing the optical properties of tissue-simulating turbid media. In the present work, using an original approach, the specific features have been revealed experimentally and interpreted physically (as owing to the polydispersity of the ensemble of scatterers) of the generally non-linear behaviour of the extinction (total attenuation) coefficient of Intralipid-20% dilutions in distilled water, depending on the Intralipid concentration, for near-infrared laser radiation of different wavelengths. At relatively low incremental concentrations, the values obtained of the extinction coefficient are shown to increase linearly being in agreement at each wavelength of concern with results for the integral scattering coefficient predicted by empiric formulae found by other authors. Comparative estimation has been performed as well, depending on the radiation wavelength, of the potentialities of single-sided optical sensing of tissue-like media. The estimates obtained outline the advantage of the wavelengths around the upper limit of the tissue optical window.     

Design and testing of a white-light, steady-state diffuse reflectance spectrometer for determination of optical properties of highly scattering systems

We present a steady-state radially resolved diffuse reflectance spectrometer capable of measuring the absorption and transport scattering spectra of tissue-simulating phantoms over an adjustable 170-nm wavelength interval in the visible and near infrared. Measurements in a variety of phantoms are demonstrated over the relevant range of tissue optical properties, and the accuracy of the instrument is found to be approximately 10% in both scattering and absorption. Monte Carlo simulations designed to test the accuracy of the instrument are presented that support the experimental findings.     

Validation study of the SeaWiFS oxygen A-band absorption correction: comparing the retrieved cloud optical thicknesses from SeaWiFS measurements

Atmospheric correction in ocean-color remote sensing corrects more than 90% of signals in the visible contributed from the atmosphere measured at satellite altitude. The Sea-viewing Wide Field-of-view Sensor (SeaWiFS) atmospheric correction uses radiances measured at two near-infrared wavelengths centered at 765 and 865 nm to estimate the atmospheric contribution and extrapolate it into the visible range. However, the SeaWiFS 765-nm band, which covers 745-785 nm, completely encompasses the oxygen A-band absorption. The O(2) A-band absorption usually reduces more than 10-15% of the measured radiance at the SeaWiFS 765-nm band. Ding and Gordon [Appl. Opt. 34, 2068-2080 (1995)] proposed a numerical scheme to remove the O(2) A-band absorption effects from the atmospheric correction. This scheme has been implemented in the SeaWiFS ocean-color imagery data-processing system. I present results that demonstrate a method to validate the SeaWiFS 765-nm O(2) A-band absorption correction by analyzing the sensor-measured radiances at 765 and 865 nm taken looking at the clouds over the oceans. SeaWiFS is usually not saturated with cloudy scenes because of its bilinear gain design. Because the optical and radiative properties of water clouds are nearly independent of the wavelengths ranging from 400 to 865 nm, the sensor-measured radiances above the cloud at the two near-infrared wavelengths are comparable. The retrieved cloud optical thicknesses from the SeaWiFS band 7 measurements are compared for cases with and without the O(2) A-band absorption corrections and from the band 8 measurements. The results show that, for air-mass values of 2-5, the current SeaWiFS O(2) A-band absorption correction works reasonably well. The validation method is potentially applicable for in-orbit relative calibration for SeaWiFS and other satellite sensors.