Quantification of fluorophore concentration in tissue-simulating media by fluorescence measurements with a single optical fiber

Quantifying fluorescent compounds in turbid media such as tissue is made difficult by the effects of multiple scattering and absorption of the excitation and emission light. The approach that we used was to measure fluorescence using a single 200-microm optical fiber as both the illumination source and the detector. Fluorescence of aluminum phthalocyanine tetrasulfonate (AlPcS4) was measured over a wide range of fluorophore concentrations and optical properties in tissue-simulating phantoms. A root-mean-square accuracy of 10.6% in AlPcS4 concentration was attainable when fluorescence was measured either interstitially or at the phantom surface. The individual effects of scattering, absorption, and the scattering phase function on the fluorescence signal were also studied by experiments and Monte Carlo simulations.     

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.     

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.     

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).     

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.     

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.     

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.     

Evaluation of optical properties of highly scattering media by moments of distributions of times of flight of photons

A novel method for the determination of the optical properties of tissue from time-domain measurements is presented. The data analysis is based on the evaluation of the first moment and the second centralized moment, i.e., the mean time of flight and the variance of the measured distribution of times of flight (DTOF) of photons injected by short (picosecond) laser pulses. Analytical expressions are derived for calculation of absorption and of reduced scattering coefficients from these moments by application of diffusion theory for infinite and semi-infinite homogeneous media. The proposed method was tested on experimental data obtained with phantoms, and results for absorption and reduced scattering coefficients obtained by the proposed method are compared with those obtained by fitting of the same data with analytical solutions of the diffusion equation. Furthermore, the accuracy of the moment analysis was investigated for a range of integration limits of the DTOF. The moment analysis may serve as a comparatively fast method for evaluating optical properties with sufficient accuracy and can be used, e.g., for on-line monitoring of optical properties of biological tissue.     

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.     

Tests of backscatter coefficient measurement using broadband pulses

An adaptation to a data reduction method is outlined for determining backscatter coefficients, eta, when broad-bandwidth pulses are employed. The accuracy of these eta values is assessed with well-characterized phantoms, for which backscatter coefficients based on their physical properties have been independently calculated. One phantom produces Rayleigh-like scattering, where the backscatter coefficient varies smoothly with frequency over the analysis bandwidth. A second phantom exhibits local maxima and minima in the scattering function versus frequency due to the presence of millimeter-sized graphite gel spheres in a gel background. The method was found to produce accurate results using time gate durations as small as 2 mus, although better accuracy is obtained for longer gate durations, particularly when the sample exhibits resonance peaks in backscatter versus frequency. Use of a Hamming window in place of a rectangular window extends the accuracy near the upper and lower limits of the frequency range.     

Design and analysis of a flow-through integrating cavity absorption meter

We present a design for a flow-through integrating cavity absorption meter. This instrument, in principle, is capable of measuring the spectral optical absorption coefficient of natural waters in situ independently of scattering effects. Monte Carlo simulations are used to determine the design parameters and evaluate instrument performance. We investigate both detector response and the distribution of radiant energy inside the instrument and present empirical equations describing these quantities as a function of the absorption coefficient. The effects of changing the instrument geometry are illustrated. Finally, we discuss the effects of scattering on the instrument performance and verify that they are negligible for natural waters.     

Experimental and numerical analysis of short-pulse laser interaction with tissue phantoms containing inhomogeneities

The objective is to perform an experimental and numerical study to analyze short-pulse laser propagation through tissue phantoms without and with inhomogeneities embedded in them. For a short-pulse laser the observed optical signal has a distinct temporal shape, and the shape is a function of the medium properties. The scattered temporal transmitted and reflected optical signals are measured experimentally with a streak camera for tissue phantoms irradiated with a short-pulse laser source. A parametric study involving different scattering and absorption coefficients of tissue phantoms and inhomogeneities, as well as the detector positions and orientations, is performed. The temporal and spatial profiles of the scattered optical signals are compared with the numerical modeling results obtained by solving the transient radiative transport equation by using the discrete ordinates technique.     

Mechanisms for attenuation in cancellous-bone-mimicking phantoms

Broadband ultrasound attenuation (BUA) in cancellous bone is useful for prediction of osteoporotic fracture risk, but its causes are not well understood. To investigate attenuation mechanisms, 9 cancellous-bone-mimicking phantoms containing nylon filaments (simulating bone trabeculae) embedded within soft-tissue-mimicking fluid (simulating marrow) were interrogated. The measurements of frequency-dependent attenuation coefficient had 3 separable components: 1) a linear (with frequency) component attributable to absorption in the soft-tissue-mimicking fluid, 2) a quasilinear (with frequency) component, which may include absorption in and longitudinal-shear mode conversion by the nylon filaments, and 3) a nonlinear (with frequency) component, which may be attributable to longitudinal-longitudinal scattering by the nylon filaments. The slope of total linear (with frequency) attenuation coefficient (sum of components #1 and #2) versus frequency was found to increase linearly with volume fraction, consistent with reported measurements on cancellous bone. Backscatter coefficient measurements in the 9 phantoms supported the claim that the nonlinear (with frequency) component of attenuation coefficient (component #3) was closely associated with longitudinal-longitudinal scattering. This work represents the first experimental separation of these 3 components of attenuation in cancellous bone-mimicking phantoms.     

Evaluation of optical coherence quantitation of analytes in turbid media by use of two wavelengths

We introduce a novel method for determining analyte concentration as a function of depth in a highly scattering media by use of a dual-wavelength optical coherence tomography system. We account for the effect of scattering on the measured attenuation by using a second wavelength that is not absorbed by the sample. We assess the applicability of this technique by measuring the concentration of water in an Intralipid phantom, using a probe wavelength of 1.53 mum and a reference wavelength of 1.31 mum. The results of our study show a strong correlation between the measured absorption and the water content of the sample. The accuracy of the technique, however, was limited by the dominance of scattering over absorption in the turbid media. Thus, although the effects of scattering were minimized, significant errors remained in the calculated absorption values. More-accurate results could be obtained with the use of more powerful superluminescent diodes and a choice of wavelengths at which absorption effects are more significant relative to scattering.     

Fiber-optic bundle design for quantitative fluorescence measurement from tissue

We demonstrate a new design for a fiber-optic bundle to measure fluorescence signals from tissue. In this design, the intensity of the signal is not significantly affected by the medium's absorption and scattering coefficients and hence depends only on the fluorophore's properties. Monte Carlo simulations of light scattering were used for designing and verifying the results obtained. The fiber-optic bundle was tested on tissue-simulating phantoms and compared with a standard nonimaging fiber-optic bundle. The new bundle was composed of 30 individual 100-mum fibers. Fibers on the end of the bundle that touch the tissue surface were separated from one another by approximately 1 mm. This design permits integration of the signal over several locations while maintaining localized sampling from regions smaller than the average mean free scattering path of the tissue. The bundle was tested by measurement of the fluorescence signals from tissue-simulating solutions containing fluorescent compounds. These studies demonstrate that the new bundle reduces the effect of the intrinsic absorption in the medium, permitting detection of fluorescence that is linearly proportional to the fluorophore concentration.     

Multiple-slice imaging of a tissue-equivalent phantom by use of time-resolved optical tomography

Following several years of development the construction of a multichannel time-resolved imaging device for medical optical tomography has been completed. Images are reconstructed from time-resolved measurements by use of a scheme that employs a finite-element diffusion-based forward model and an iterative reconstruction solver. Prior to testing on clinical subjects the fully automated instrument and the reconstruction software are evaluated with tissue-equivalent phantoms. We describe our first attempt to generate multiple-slice images of a phantom without uniform properties along the axial direction, while still using a computationally fast two-dimensional reconstruction algorithm. The image quality is improved by the employment of an approximate correction method that uses scaling factors derived from the ratios of finite-element forward simulations in two and three spatial dimensions. The 32-channel system was employed to generate maps of the internal scattering and the absorption properties at 14 different transverse planes across the phantom. The images clearly reveal the locations of small inhomogeneous regions embedded within the phantom. These results were obtained by use of purely temporal data and without resource to reference measurements.     

Experimental test of a perturbation model for time-resolved imaging in diffusive media

We report what to our knowledge is a novel perturbation approach for time-resolved transmittance imaging in diffusive media, based on the diffusion approximation with extrapolated boundary conditions. The model relies on the method of Padé approximants and consists of a nonlinear approximation of time-resolved transmittance curves in the presence of an inclusion. The proposed model is intended to extend the range of applicability of perturbation models when applied to inclusions that are non-point-like. We test the model on different tissue phantoms with scattering only, absorbing only, and both scattering and absorbing inclusions. Maps of the optical properties are displayed, and the results are compared with those obtained by means of the usual linear approximation of time-resolved transmittance curves. We found that the nonlinear approach gives a better prediction for absolute values of the scattering and absorption coefficients of inclusions, when the inclusion optical properties are higher than the surrounding background. Furthermore, better-resolved spots and a reduced cross talk between the two parameters are found in the reconstructed maps. Because the range of the optical properties spanned by the considered phantoms covers the values expected for optical mammography, the application of the reported reconstruction method to in vivo images of a breast appears promising from a diagnostic viewpoint.     

Streak camera: a multidetector for diffuse optical tomography

We describe an experimental setup for time-resolved diffuse optical tomography that uses a seven-channel light guide to transmit scattered light to a streak camera. This setup permits the simultaneous measurement of the time profiles of photons reemitted at different boundary sites of the objects studied. The instrument, its main specifications, and detector-specific data analysis before image reconstruction are described. The instrumentation was tested with phantoms simulating biological tissue, and the absorption and reduced scattering images that were obtained are discussed.     

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.