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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-[micro]m optical fiber as both the illumination source and the detector. Fluorescence of aluminum phthalocyanine tetrasulfonate (AlPc[S.sub.4]) 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 AlPc[S.sub.4] 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. OCIS codes: 170.3890, 170.6280, 170.5180, 170.7050.

Measurement of the concentration of fluorescent compounds in turbid media is difficult because the absorption and multiple scattering of excitation and emission of light has a large effect on the detected fluorescence. For surface measurements with optical fibers we demonstrate by experiments and numerical simulation that this effect can be minimized by measurement of the fluorescence at one source--detector distance, the diffusely reflected excitation light at a second distance, and with the ratio of these two signals as an indicator of fluorophore concentration. For optical properties typical of soft tissue in the red and the near infrared the optimum performance is obtained by measurement of fluorescence at 0.65 mm and reflectance at 1.35 mm. This choice reduces the rms error in fluorophore concentration to 14.6% over a wide range of absorption and scattering coefficients. OCIS codes: 170.3890, 170.6280, 170.5180, 170.7050.

We have examined the possibility of determining the optical properties of a two-layer medium by using a diffusion approximation radiation transport model [Appl. Opt. 37, 779 (1998)]. Continuous-wave and frequency-domain (FD) low-noise Monte Carlo (MC) data were fitted to the model. Marquardt-Levenberg and a simulated annealing algorithm were used and compared as optimization techniques. Our particular choice of optical properties for the two-layer model was consistent with skin and underlying fat in the presence of an exogenous chromophore [Appl. Opt. 37, 1958 (1998)]. The results are therefore specific to this set of optical properties. It was found that the cw diffusion solution could never be used to estimate all optical properties reliably. The combined cw and FD solutions could not be used to estimate some of the top-layer optical properties to an accuracy of better than 10%, although the absorption and the transport scattering coefficients of the bottom layer could be estimated to within 7% and 0.5%, respectively. No improvement was found from simultaneously fitting MC data at three different modulation frequencies. These results point to the need for a more accurate radiation transfer model at small source-detector separations.

Most instruments used to measure tissue optical properties noninvasively employ data-analysis algorithms that rely on the simplifying assumption that the tissue is semi-infinite and homogeneous. The influence of a layered tissue architecture on the determination of the scattering and absorption coefficients has been investigated in this study. Reflectance as a function of distance from a point source for a two-layered tissue architecture that simulates skin overlying fat was calculated by using a Monte Carlo code. These data were analyzed by using a diffusion theory model for a homogeneous semi-infinite medium to calculate the scatter and absorption coefficients. Depending on the algorithm and the radial distance, the estimated tissue optical properties were different from those of either layer, and under some circumstances, physically impossible. In addition, the sensitivity and cross talk of the estimated optical properties to changes in input optical properties were calculated for different layered geometries. For typical optical properties of skin, the sensitivity to changes in optical properties is highly dependent on the layered architecture, the measurement distance, and the fitting algorithm. Furthermore, a change in the input absorption coefficient may result in an apparent change in the measured scatter coefficient, and a change in the input scatter coefficient may result in an apparent change in the measured absorption coefficient.

Light propagation in two-layered turbid media having an infinitely thick second layer is investigated in the steady-state, frequency, and time domains. A solution of the diffusion approximation to the transport equation is derived by employing the extrapolated boundary condition. We compare the reflectance calculated from this solution with that computed with Monte Carlo simulations and show good agreement. To investigate if it is possible to determine the optical coefficients of the two layers and the thickness of the first layer, the solution of the diffusion equation is fitted to reflectance data obtained from both the diffusion equation and the Monte Carlo simulations. Although it is found that it is, in principle, possible to derive the optical coefficients of the two layers and the thickness of the first layer, we concentrate on the determination of the optical coefficients, knowing the thickness of the first layer. In the frequency domain, for example, it is shown that it is sufficient to make relative measurements of the phase and the steady-state reflectance at three distances from the illumination point to obtain useful estimates of the optical coefficients. Measurements of the absolute steady-state spatially resolved reflectance performed on two-layered solid phantoms confirm the theoretical results. OCIS codes: 290.7050, 050.1960, 170.6930.

A dosimetric system has been developed to measure the spatially resolved light dose absorbed by a photosensitizer in a tissue-simulating medium. These gelatin-based dosimeters had macroscopic optical scattering and absorption properties that are typical for homogeneous tissue and contained the photosensitizer benzoporphyrin derivative monoacid (BPD-MA). A reporter molecule, 2[prime]7[prime]-dichlorofluorescin diacetate (DCF-DA), served as an actinometer, which could be photosensitized by BPD-MA to generate a highly fluorescent photoproduct. The relative photosensitizing efficiencies of high-intensity pulsed and cw laser light were compared in these tissue-simulating dosimeters. These measurements demonstrate an increase in penetration for pulsed light as compared with cw light in the dosimeters. A numerical simulation of the light propagation based on optical diffusion theory was used along with the energy levels of the photosensitizer molecule to examine the mechanisms involved in the absorbed dose. The increased penetration of high-intensity pulsed light was due to a transient decrease in the absorption of the photosensitizer, resulting from saturation of the photosensitizer optical transitions. This study provides the first direct comparison of the photodynamic dose absorbed by a photosensitizer using both high-intensity pulsed and cw laser light in a tissue-simulating medium. These measurements demonstrate that a small increase in depth of treatment is possible with pulsed laser light as compared with cw laser light simply on the basis of the unique photochemistry of the photosensitizer. However, this effect still needs to be examined carefully in tumor tissue, where other biological or chemical effects may become significant. Key words: Photodynamic therapy, photosensitizer, benzoporphyrin derivative, tissue optical property, tissue phantom.

An experimental study of the detectability of an object embedded in optically tissue-equivalent media by frequency-domain image reconstruction is presented. The experiments were performed in an 86-mm-diameter cylindrical phantom containing an optically homogeneous cylindrical target whose absorption and scattering properties presented a 2:1 contrast with the background medium. The parameter space explored during experimentation involved object size (15-, 8-, and 4-mm targets) and location (centered, 20-mm off-centered, and 35-mm off-centered) variations. Image reconstruction was achieved with a previously reported regularized least-squares approach that incorporates finite-element solutions of the diffusion equation and Newton's method solutions of the nonlinear minimization problem. Also included during image formation were image enhancement schemes - (1) total variation minimization, (2) dual meshing, and (3) spatial low-pass filtering - which have recently been added. Quantitative measures of image quality including the size, location, and shape of the heterogeneity along with errors in its recovered optical property values are used to quantify the image reconstructions. The results show that a near 22:1 ratio of tissue thickness relative to detectable object size has been achieved with this approach in the laboratory conditions and parameter space that have been investigated.

Laser Doppler measurements were performed on scattering liquids flowing through a highly scattering static medium to determine the scattering coefficient and the anisotropy factor of the liquids. Monte Carlo simulations of light propagation in the static and moving media were used to calculate the Doppler spectra for suspensions of polystyrene spheres in water, and these spectra were in excellent agreement with experimental results. A faster Monte Carlo code was developed so that nonlinear regressions to the measured laser Doppler spectra could be used to determine the anisotropy factor of other liquids. This approach was used to deduce the scattering properties of Intralipid and blood at [Lambda] = 820 nm. It was found that the anisotropy factor of blood is well described by Mie theory in contradiction to results reported in the literature that were obtained by goniometric measurement of the phase function. Key words: Laser Doppler, Monte Carlo, anisotropy factor, blood.

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.sup.-1] and absorption coefficients between 0.002 and 0.1 [mm.sup.-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. Key words: Tissue optics, reflectance, Monte Carlo, neural network.

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.

A diffusion model of noninvasive absorption spectroscopy was used to determine how the change in signal resulting from a point absorber depends on the position of that absorber relative to the source and detector. This is equivalent to calculating the relative probability that a photon will visit a certain location in tissue before its detection. Experimental mapping of the point-target response in tissue-simulating materials confirmed the accuracy of the model. For steady-state spectroscopy a simple relation was derived between the mean depth visited by detected photons, the source-detector separation, and the optical penetration depth. It was also demonstrated theoretically that combining a pulsed source with time-gated detection provides additional control over the spatial distribution of the photon-visit probability. Key words: Absorption spectroscopy, tissue optics, diffusion theory.

In general it is not possible to write an analytic expression for the fluorescence signal generated by a fluorophore distributed in a scattering medium such as tissue. However, by assuming that the scattering properties of the tissue are the same at the excitation and emission wavelengths, we have derived a simple relation between the fluorescence and the scatter signals. Along with diffusion theory, this was used to write expressions for the fluorescence signal detected at the tissue surface in both the time and the frequency domains. Experiments using the fluorophore aluminum chlorosulfonated phthalocyanine in tissue-simulating materials confirmed the accuracy of the model. Applications to in vivo spectroscopy are discussed.

When a picosecond light pulse is incident upon a turbid medium such as tissue, the temporal distribution of diffusely reflected and transmitted photons depends on the optical absorption and scattering properties of the medium. From diffusion theory it is possible to derive analytic expressions for the pulse shape in terms of the optical interaction coefficients of a homogeneous semi-infinite medium. Experimental tests of this simple model in tissue-simulating liquid phantoms of different geometries are presented here. The results of these tests show that, in a semi-infinite phantom, the application of the diffusion model provides estimates of the absorption and transport-scattering coefficients that are accurate to better than 10%. Comparable accuracy was also obtained with this simple model for finite slab, cylindrical, and spherical volumes as long as the objects were of sufficient size. For smaller volumes the absorption coefficient was overestimated because of the significant loss of photons at the boundaries of the object.

The general two-layer inverse problem in biomedical photon migration is to estimate the absorption and scattering coefficients of each layer as well as the top-layer thickness. We attempted to solve this problem, using experimental and simulated spatially resolved frequency-domain (FD) reflectance for optical properties typical of skin overlying muscle or skin overlying fat in the near infrared. Two forward models of light propagation were used: a two-layer diffusion solution [Appl. Opt. 37, 779 (1998)] and a hybrid Monte Carlo (MC) diffusion model [Appl. Opt. 37, 7401 (1998)]. MC-simulated FD reflectance data were fitted as relative measurements to the hybrid and the pure diffusion models. It was found that the hybrid model could determine all the optical properties of the two-layer media studied to ~5%. Also, the same accuracy could be achieved by means of fitting MC-simulated cw reflectance data as absolute measurements, but fitting them as relative ones is an ill-posed problem. In contrast, two-layer diffusion could not retrieve the top-layer optical properties as accurately for FD data and was ill-posed for both relative and absolute cw data. The hybrid and the pure diffusion models were also fitted to experimental FD reflectance measurements from two-layer tissue-simulating phantoms representative of skin-on-fat and skin-on-muscle baseline optical properties. Both the hybrid and the diffusion models could determine the optical properties of the lower layer. The hybrid model demonstrated its potential to retrieve quantitatively the transport scattering coefficient of skin (the upper layer), which was not possible with the pure diffusion model. Systematic discrepancies between model and experiment may compromise the accuracy of the deduced top-layer optical properties. Identifying and eliminating such discrepancies is critical to practical application of the method. OCIS codes: 170.5280, 290.1990, 290.7050, 170.4090.

We propose a hybrid Monte Carlo (MC) diffusion model for calculating the spatially resolved reflectance amplitude and phase delay resulting from an intensity-modulated pencil beam vertically incident on a two-layer turbid medium. The model combines the accuracy of MC at radial distances near the incident beam with the computational efficiency afforded by a diffusion calculation at further distances. This results in a single forward calculation several hundred times faster than pure MC, depending primarily on model parameters. Model predictions are compared with MC data for two cases that span the extremes of physiologically relevant optical properties: skin overlying fat and skin overlying muscle, both in the presence of an exogenous absorber. It is shown that good agreement can be achieved for radial distances from 0.5 to 20 mm in both cases. However, in the skin-on-muscle case the choice of model parameters and the definition of the diffusion coefficient can lead to some interesting discrepancies. [C] 2000 Optical Society of America OCIS codes: 170.5280, 290.1990.