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).     

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.     

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.     

Recovery of hemoglobin oxygen saturation and intrinsic fluorescence with a forward-adjoint model

We present two forward-adjoint models for recovering intrinsic fluorescence spectra and hemoglobin oxygen saturation of turbid samples. The first fits measured diffuse reflectance spectra to obtain the absorption and scattering spectra of the medium, and these are then used to correct distortions imposed on the fluorescence spectrum by absorption and scattering. The second fits only the measured fluorescence spectrum to determine simultaneously the amplitudes of absorption and fluorescence basis spectra and scattering parameters. Both methods are validated with Monte Carlo simulations and experimentally in scattering phantoms containing nicotinamide adenine dinucleotide and human erythrocytes. Preliminary measurements from murine tumors in vivo are presented.     

Fiber-optic probe for noninvasive real-time determination of tissue optical properties at multiple wavelengths

We present a compact, fast, and versatile fiber-optic probe system for real-time determination of tissue optical properties from spatially resolved continuous-wave diffuse reflectance measurements. The system collects one set of reflectance data from six source-detector distances at four arbitrary wavelengths with a maximum overall sampling rate of 100 Hz. Multivariate calibration techniques based on two-dimensional polynomial fitting are employed to extract and display the absorption and reduced scattering coefficients in real-time mode. The four wavelengths of the current configuration are 660, 785, 805, and 974 nm, respectively. Cross-validation tests on a 6 x 7 calibration matrix of Intralipid-dye phantoms showed that the mean prediction error at, e.g., 785 nm was 2.8% for the absorption coefficient and 1.3% for the reduced scattering coefficient. The errors are relative to the range of the optical properties of the phantoms at 785 nm, which were 0-0.3/cm for the absorption coefficient and 6-16/cm for the reduced scattering coefficient. Finally, we also present and discuss results from preliminary skin tissue measurements.     

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.     

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.     

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.     

Sequential estimation of optical properties of a two-layered epithelial tissue model from depth-resolved ultraviolet-visible diffuse reflectance spectra

A method for estimating the optical properties of two-layered media (such as squamous epithelial tissue) over a range of wavelengths in the ultraviolet-visible spectrum is proposed and tested with Monte Carlo modeling. The method first used a fiber-optic probe with angled illumination and the collection fibers placed at a small separation (or=1000 microm) was used to detect diffuse reflectance preferentially from the bottom layer. A second Monte Carlo-based inverse model for a two-layered medium was applied to estimate the bottom layer optical properties, as well as the top layer thickness, given that the top layer optical properties have been estimated. The results of Monte Carlo validation show that this method works well for an epithelial tissue model with a top layer thickness ranging from 200 to 500 microm. For most thicknesses within this range, the absorption coefficients were estimated to within 15% of the true values, the reduced scattering coefficients were estimated to within 20% and the top layer thicknesses were estimated to within 20%. The application of a variance reduction technique to the Monte Carlo modeling proved to be effective in improving the accuracy with which the optical properties are estimated.     

Characterization of spatial and temporal variations in the optical properties of tissuelike media with diffuse reflectance imaging

We describe a method to characterize spatial or temporal changes in the optical properties of turbid media using diffuse reflectance images acquired under broad-beam illumination conditions. We performed experiments on liquid phantoms whose absorption (mu(a)) and reduced scattering (mu(s)') coefficients were representative of those of biological tissues in the near infrared. We found that the relative diffuse reflectance R depends on mu(a) and mu(s)' only through the ratio mu(a)/mu(s)' and that dependence can be well described with an analytical expression previously reported in the literature [S. L. Jacques, Kluwer Academic Dordrecht (1996)]. We have found that this expression for R deviates from experimental values by no more than 8% for various illumination and detection angles within the range 0 degrees-30 degrees. Therefore, this analytical expression for R holds with good approximation even if the investigated medium presents curved or irregular surfaces. Using this expression, it is possible to translate spatial or temporal changes in the relative diffuse reflectance from a turbid medium into quantitative estimates of the corresponding changes of (mu(a)/mu(s)')(1/2). In the case of media with optical properties similar to those of tissue in the near infrared, we found that the changes mu(a)/mu(s)' should occur over a volume approximately 2 mm deep and 4 mm x 4 mm wide to apply this expression.     

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.     

Portable, Fiber-Based, Diffuse Reflection Spectroscopy (DRS) Systems for Estimating Tissue Optical Properties

Steady-state diffuse reflection spectroscopy is a well-studied optical technique that can provide a noninvasive and quantitative method for characterizing the absorption and scattering properties of biological tissues. Here, we compare three fiber-based diffuse reflection spectroscopy systems that were assembled to create a light-weight, portable, and robust optical spectrometer that could be easily translated for repeated and reliable use in mobile settings. The three systems were built using a broadband light source and a compact, commercially available spectrograph. We tested two different light sources and two spectrographs (manufactured by two different vendors). The assembled systems were characterized by their signal-to-noise ratios, the source-intensity drifts, and detector linearity. We quantified the performance of these instruments in extracting optical properties from diffuse reflectance spectra in tissue-mimicking liquid phantoms with well-controlled optical absorption and scattering coefficients. We show that all assembled systems were able to extract the optical absorption and scattering properties with errors less than 10%, while providing greater than ten-fold decrease in footprint and cost (relative to a previously well-characterized and widely used commercial system). Finally, we demonstrate the use of these small systems to measure optical biomarkers in vivo in a small-animal model cancer therapy study. We show that optical measurements from the simple portable system provide estimates of tumor oxygen saturation similar to those detected using the commercial system in murine tumor models of head and neck cancer.     

Monte Carlo-based inverse model for calculating tissue optical properties. Part II: Application to breast cancer diagnosis

The Monte Carlo-based inverse model of diffuse reflectance described in part I of this pair of companion papers was applied to the diffuse reflectance spectra of a set of 17 malignant and 24 normal-benign ex vivo human breast tissue samples. This model allows extraction of physically meaningful tissue parameters, which include the concentration of absorbers and the size and density of scatterers present in tissue. It was assumed that intrinsic absorption could be attributed to oxygenated and deoxygenated hemoglobin and beta-carotene, that scattering could be modeled by spheres of a uniform size distribution, and that the refractive indices of the spheres and the surrounding medium are known. The tissue diffuse reflectance spectra were evaluated over a wavelength range of 400-600 nm. The extracted parameters that showed the statistically most significant differences between malignant and nonmalignant breast tissues were hemoglobin saturation and the mean reduced scattering coefficient. Malignant tissues showed decreased hemoglobin saturation and an increased mean reduced scattering coefficient compared with nonmalignant tissues. A support vector machine classification algorithm was then used to classify a sample as malignant or nonmalignant based on these two extracted parameters and produced a cross-validated sensitivity and specificity of 82% and 92%, respectively.     

Changes in optical properties of rat cerebral cortical slices during oxygen glucose deprivation

We simultaneously measured the diffuse reflectance spectra and transmittance spectra of in vitro rat cerebral cortical tissue slices perfused with artificial cerebrospinal fluid (aCSF) in the wavelength range from 500 to 900 nm. An ischemia-like condition in the cortical tissue was induced by oxygen/glucose deprivation (OGD) of the aCSF. Diffuse reflectance and transmittance of the cortical slices were decreased and increased, respectively, during OGD. Spectral data of reduced scattering coefficients and absorption coefficients were estimated by the inverse Monte Carlo simulation for light transport in tissue. As with OGD, significant decrease of the reduced scattering coefficients and alteration of the absorption coefficient spectrum were observed over the measured wavelength range. The mean maximum amplitudes of change in the absorption coefficient at 520, 550, 605, and 830 nm were 0.33 ± 0.14, 0.30 ± 0.12, 0.30 ± 0.14, and -0.04 ± 0.16, respectively, whereas those in the reduced scattering coefficient at 520, 550, 605, and 830 nm were -0.37 ± 0.08, -0.38 ± 0.08, -0.38 ± 0.08, and -0.39 ± 0.08. Variations in the reduced scattering coefficients implied cell deformation mainly due to cell swelling, whereas those in the absorption spectra indicated reductions in heme aa(3) and CuA in cytochrome c oxidase and cytochrome c.     

A feature parameter of optical diffuse reflectance on normal and malignant human breast tissue

Light that is delivered to the tissue surface undergoes multiple elastic scattering and absorption; part of it returns to the surface as optical diffuse reflectance, carrying quantitative information on the structure and composition of the measured tissue. In this paper, we utilized a well tested and publicly available Monte Carlo simulation software to simulate optical diffuse reflectance on normal and malignant human breast tissue in the visible wavelength region 450–650 nm. Based on the Monte Carlo simulation results, we discovered a feature parameter of optical diffuse reflectance on the simulated tissue surface. Normal and malignant human breast tissue may be discriminated by the value of the feature parameter. The values of the feature parameter are shown for normal and malignant human breast tissue in the visible wavelength region 450–650 nm. Experiments with tissue phantoms showed that the feature parameter is correlated to the component of the phantom. So the feature parameter is useful for the non-invasive optical diagnosis of biological tissue.     

Influence of a superficial layer in the quantitative spectroscopic study of strongly scattering media

We have experimentally investigated the meaning of the effective optical absorption [mu(a)((eff))] and the reduced scattering [mu(s)?((eff))] coefficients measured on the surfaces of two-layered turbid media, using the diffusion equation for homogeneous, semi-infinite media. We performed frequency-domain spectroscopy in a reflectance geometry, using source-detector distances in the range 1.5-4.5 cm. We measured 100 samples, each made of one layer (thickness in the range 0.08-1.6 cm) on top of one semi-infinite block. The optical properties of the samples were similar to those of soft tissues in the near infrared. We found that the measured effective optical coefficients are representative of the underlying block if the superficial layer is less than ~0.4 cm thick, whereas they are representative of the superficial layer if it is more than ~1.3 cm thick.     

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.     

Scaling method for fast Monte Carlo simulation of diffuse reflectance spectra from multilayered turbid media

A scaling Monte Carlo method has been developed to calculate diffuse reflectance from multilayered media with a wide range of optical properties in the ultraviolet-visible wavelength range. This multilayered scaling method employs the photon trajectory information generated from a single baseline Monte Carlo simulation of a homogeneous medium to scale the exit distance and exit weight of photons for a new set of optical properties in the multilayered medium. The scaling method is particularly suited to simulating diffuse reflectance spectra or creating a Monte Carlo database to extract optical properties of layered media, both of which are demonstrated in this paper. Particularly, it was found that the root-mean-square error (RMSE) between scaled diffuse reflectance, for which the anisotropy factor and refractive index in the baseline simulation were, respectively, 0.9 and 1.338, and independently simulated diffuse reflectance was less than or equal to 5% for source-detector separations from 200 to 1500 microm when the anisotropy factor of the top layer in a two-layered epithelial tissue model was varied from 0.8 to 0.99; in contrast, the RMSE was always less than 5% for all separations (from 0 to 1500 microm) when the anisotropy factor of the bottom layer was varied from 0.7 to 0.99. When the refractive index of either layer in the two-layered tissue model was varied from 1.3 to 1.4, the RMSE was less than 10%. The scaling method can reduce computation time by more than 2 orders of magnitude compared with independent Monte Carlo simulations.     

Determination of tissue optical properties from diffuse reflectance profiles by multivariate calibration

We describe a method for determining the reduced scattering and absorption coefficients of turbid biological media from the spatially resolved diffuse reflectance. A Sugeno Fuzzy Inference System in conjunction with data preprocessing techniques is employed to perform multivariate calibration and prediction on reflectance data generated by Monte Carlo simulations. The preprocessing consists of either a principal component analysis or a new, extended curve-fitting procedure originating from diffusion theory. Prediction tests on reflectance data with absorption coefficients between 0.04 and 0.06 mm(-1) and reduced scattering coefficients between 0.45 and 0.99 mm(-1) show the root-mean-square error of this method to be 0.25% for both coefficients. With reference to practical applications, we also describe how the prediction accuracy is affected by using relative instead of absolute reflectance data, by imposing measurement noise on the reflectance data, and by changing the number and the position of detectors.     

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.