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.     

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.     

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.     

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 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.     

Optical property measurements of turbid media in a small-volume cuvette with frequency-domain photon migration

A frequency-domain photon migration (FDPM) technique is developed for quantitative measurement of the absorption and reduced scattering coefficients of highly turbid samples in a small-volume (0.45-ml) reflective cuvette. We present both an analytical model for the FDPM cuvette and its experimental verification, using calibrated phantoms and suspensions of living cells. FDPM model fits to experimental data demonstrate that the reduced scattering (mu(s)?) and absorption (mu(a)) coefficients can be derived with accuracies of 5-10% and 10-15%, respectively. Changing the cuvette wall reflectivity alters the frequency-dependent behavior of photon density waves (PDWs). For highly reflective wall boundaries (R(eff) >/= 90-95%), PDW confinement leads to substantial enhancement in both amplitude and phase compared with identical samples in infinite media. Results from experiments on microsphere suspensions are compared with predictions from Mie theory to assess the potential of this method to interpret scattering properties in terms of scatterer size and density. Optical property measurements of biological cell suspensions are reported, and the possibility of optically monitoring cell physiology in a carefully controlled environment is demonstrated.     

Effect of surface roughness on determination of tissue optical properties obtained by diffusion approximation

A nylon bar with different surface roughness is used as a simulation sample of biological tissue for the determination of optical properties by using the spatially resolved steady-state diffuse reflection technique. The results obtained indicate that surface roughness has some effects on the determination of the optical properties of the nylon bar. The determined reduced scattering coefficient decreases with the decrease of the surface roughness of the nylon bar and goes to a constant for the lower surface roughness, and the determined absorption coefficient increases with the decrease of the surface roughness of the nylon bar. Consequently, the optical properties of the tissues obtained by the spatially resolved steady-state diffuse reflection technique should be modified.     

Application of frequency domain photon migration to particle size analysis and monitoring of pharmaceutical powders

The frequency domain photon migration (FDPM) technique was employed to determine mean particle size of pharmaceutical powders. Results show that the FDPM-measured scattering coefficient increases linearly with reciprocal mean particle size of powdered samples. In contrast to near-infrared spectroscopy techniques, FDPM technique enables determination of scattering and absorption separately so that it does not require data pretreatment and chemometric calibration models. In addition, this unique advantage provides more detailed information about powder samples, which can be used as a potential tool for on-line monitoring of not only variation of active pharmaceutical ingredient concentrations from changes in the absorption coefficient but also variation of particle sizes from changes in the scattering coefficient.     

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.     

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.     

Characterization of pigment particle absorption efficiencies using frequency domain photon migration

Time-dependent measurements of multiply scattered light were made using frequency domain photon migration (FDPM) techniques in polystyrene latex as a function of ppm pigment concentration (by weight) in order to determine the wavelength-dependent absorption efficiencies for three different pigment particles. The results demonstrate that the absorption spectra of pigment particles within their dispersing vehicles concur with the complementary color chart. FDPM offers a first-principles method for assessing optical characteristics of pigments within their dispersing vehicles and without the need to resort to conventional measurement of diffuse reflectance from coatings and data analysis using phenomenological theory.     

Quantifying the absorption and reduced scattering coefficients of tissuelike turbid media over a broad spectral range with noncontact Fourier-transform hyperspectral imaging

Absorption (mu(a)) and reduced scattering (mu(s)') spectra of turbid media were quantified with a noncontact imaging approach based on a Fourier-transform interferometric imaging system (FTIIS). The FTIIS was used to collect hyperspectral images of the steady-state diffuse reflectance from turbid media. Spatially resolved reflectance data from Monte Carlo simulations were fitted to the recorded hyperspectral images to quantify mu(a) and mu(s)' spectra in the 550-850-nm region. A simple and effective calibration approach was introduced to account for the instrument response. With reflectance data that were close to and far from the source (0.5-6.5 mm), mu(a) and mu(s)' of homogeneous, semi-infinite turbid phantoms with optical property ranges comparable with those of tissues were determined with an accuracy of +/-7% and +/-3%, respectively. Prediction accuracy for mu(a) and mu(s)' degraded to +/-12% and +/-4%, respectively, when only reflectance data close to the source (0.5-2.5 mm) were used. Results indicate that reflectance data close to and far from the source are necessary for optimal quantification of mu(a) and mu(s)'. The spectral properties of mu(a) and mu(s)' values were used to determine the concentrations of absorbers and scatterers, respectively. Absorber and scatterer concentrations of two-chromophore turbid media were determined with an accuracy of +/-5% and +/-3%, respectively.     

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.     

High-Transmission-Efficiency and Side-Viewing Micro OIDRS Probe for Fast and Minimally-Invasive Tumor Margin Detection

The determination of a cancer free margin I organs is a difficult and time consuming process, with an unmet need for rapid determination of tumor margin at surgery. In this paper, we report the design, fabrication and testing of a novel miniaturized optical sensor probe with "side-viewing" capability. Its unprecedented small size, unique "side-viewing" capability and high optical transmission efficiency enable the agile maneuvering and efficient data collection even in the narrow cavities inside the human body. The sensor probe consists of four micromachined substrates with optical fibers for oblique light incidence and collection of spatially resolved diffuse reflectance from the contacted tissues. The optical sensor probe has been used to conduct the oblique incidence diffuse reflectance spectroscopy (OIDRS) on a human pancreatic specimen. Based on the measurement results, the margin of the malignant tumor has been successfully determined optically, which matches well with the histological results.     

Experimental determination of photon propagation in highly absorbing and scattering media

Optical imaging and tomography in tissues can facilitate the quantitative study of several important chromophores and fluorophores. Several theoretical models have been validated for diffuse photon propagation in highly scattering and low-absorbing media that describe the optical appearance of tissues in the near-infrared (NIR) region. However, these models are not generally applicable to quantitative optical investigations in the visible because of the significantly higher tissue absorption in this spectral region compared with that in the NIR. We performed photon measurements through highly scattering and absorbing media for ratios of the absorption coefficient to the reduced scattering coefficient ranging approximately from zero to one. We examined experimentally the performance of the absorption-dependent diffusion coefficient defined by Aronson and Corngold [J. Opt. Soc. Am. A 16, 1066 (1999)] for quantitative estimations of photon propagation in the low- and high-absorption regimes. Through steady-state measurements we verified that the transmitted intensity is well described by the diffusion equation by considering a modified diffusion coefficient with a nonlinear dependence on the absorption. This study confirms that simple analytical solutions based on the diffusion approximation are suitable even for high-absorption regimes and shows that diffusion-approximation-based models are valid for quantitative measurements and tomographic imaging of tissues in the visible.     

Simultaneous extraction of optical transport parameters and intrinsic fluorescence of tissue mimicking model media using a spatially resolved fluorescence technique

We present a method based on spatially resolved fluorescence measurement for the simultaneous estimation of optical transport parameters, namely, the reduced scattering coefficient (micro s'), the absorption coefficient (micro a), and the intrinsic fluorescence spectra from turbid media. The accuracy of this approach was tested by conducting studies on a series of tissue-simulating phantoms with known optical transport properties. The estimated relative error in the values for micro s' and micro a using this technique was found to be < or =10%. Furthermore, the line shape and intensity of the intrinsic fluorescence recovered by using this approach were observed to be free from the distorting effects of the wavelength-dependent absorption and scattering properties of the medium, and they were in excellent agreement with the directly measured intrinsic fluorescence spectra of the fluorophores.     

Validating the assumption to the interference approximation by use of measurements of absorption efficiency and hindered scattering in dense suspensions

Frequency domain photon migration (FDPM) measurements were employed to accurately quantify optical properties of both the suspending fluid and particles within dense polystyrene suspensions of 143- or 226-nm mean diameter at varying concentrations (5-30% by volume). The measured absorption coefficients varied linearly with particle volume fraction whereas the isotropic scattering coefficients varied nonlinearly in agreement with the prediction that utilizes the hard-sphere structure factor model. These results validate the interference approximation of light scattering to describe light propagation accurately within dense suspensions. Furthermore, owing to the accuracy of FDPM absorption measurements, the imaginary refractive indices for both particles and their suspending fluid were determined and were found to compare favorably with literature values.     

Why do veins appear blue? A new look at an old question

We investigate why vessels that contain blood, which has a red or a dark red color, may look bluish in human tissue. A CCD camera was used to make images of diffusely reflected light at different wavelengths. Measurements of reflectance that are due to model blood vessels in scattering media and of human skin containing a prominent vein are presented. Monte Carlo simulations were used to calculate the spatially resolved diffuse reflectance for both situations. We show that the color of blood vessels is determined by the following factors: (i) the scattering and absorption characteristics of skin at different wavelengths, (ii) the oxygenation state of blood, which affects its absorption properties, (iii) the diameter and the depth of the vessels, and (iv) the visual perception process.     

Three-dimensional optical tomography: resolution in small-object imaging

Near-infrared (NIR) optical tomography provide estimates of the internal distribution of optical absorption and transport scattering from boundary measurement of light propagation within biological tissue. Although this is a truly three-dimensional (3D) imaging problem, most research to date has concentrated on two-dimensional modeling and image reconstruction. More recently, 3D imaging algorithms are demonstrating better estimation of the light propagation within the imaging region and are providing the basis of more accurate image construction algorithms. As 3D methods emerge, it will become increasingly important to evaluate their resolution, contrast, and localization of optical property heterogeneity. We present a concise study of 3D reconstructed resolution of a small, low-contrast, absorbing and scattering anomaly as it is placed in different locations within a cylindrical phantom. The object is an 8-mm-diameter cylinder, which represents a typical small target that needs to be resolved in NIR mammographic imaging. The best resolution and contrast is observed when the object is located near the periphery of the imaging region (12-22 mm from the edge) and is also positioned within the multiple measurement planes, with the most accurate results seen for the scatter image when the anomaly is at 17 mm from the edge. Furthermore, the accuracy of quantitative imaging is increased to almost 100% of the target values when a priori information regarding the internal structure of imaging domain is utilized.