Quantifying the properties of two-layer turbid media with frequency-domain diffuse reflectance

Noncontact, frequency-domain measurements of diffusely reflected light are used to quantify optical properties of two-layer tissuelike turbid media. The irradiating source is a sinusoidal intensity-modulated plane wave, with modulation frequencies ranging from 10 to 1500 MHz. Frequency-dependent phase and amplitude of diffusely reflected photon density waves are simultaneously fitted to a diffusion-based two-layer model to quantify absorption (mu(a)) and reduced scattering (mu(s)') parameters of each layer as well as the upper-layer thickness (l). Study results indicate that the optical properties of two-layer media can be determined with a percent accuracy of the order of +/-9% and +/-5% for mu(a) and mu(s)', respectively. The accuracy of upper-layer thickness (l) estimation is as good as +/-6% when optical properties of upper and lower layers are known. Optical property and layer thickness prediction accuracy degrade significantly when more than three free parameters are extracted from data fits. Problems with convergence are encountered when all five free parameters (mu(a) and mu(s)' of upper and lower layers and thickness l) must be deduced.     

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.     

Estimation of optical properties of turbid media: experimental comparison of spatially and temporally resolved reflectance methods

We compare two methods for the optical characterization of turbid media. The estimates of the absorption and reduced scattering coefficients (mu(a) and mu(')(s)) by a spatially resolved method and a time-resolved method are performed on tissue-like phantoms. Aqueous suspension of microspheres and Intralipid are used as scattering media with the addition of ink as an absorber. mu(')(s) is first measured on weakly absorbing media. The robustness of these measurements is then tested with respect to a variation of mu(a). The spatially resolved method gave more accurate estimates for mu(')(s) whereas the time-resolved method gave better results for mu(a) estimates.     

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.     

Fractal dimension analysis of time-resolved diffusely scattered light from turbid samples

To improve quantification of optical properties in highly scattering and absorbing samples, time-correlated single photon counting measurements were analyzed using quantities related to the correlation dimension. Photon time-of-flight (TOF) distributions were collected in reflection and transmission optical configurations from samples made of cream and water-soluble dye (0 < mu(a) < 0.05 mm(-1); 100 < mu(s) < 250 mm(-1)). It was found that absorption and scattering properties of samples could be accurately quantified from information used to determine the correlation dimension. Scattering coefficients were estimated with less than 4% error for both optical configurations. Absorption estimates were made with CVs of 7.5 and 9.6% for reflection and transmission, respectively. Overall, fractal dimension analysis of TOF distributions provides a simple method of determining the optical properties of a sample.     

Multiple scattering of a photon flux: implications for the integral average cosine of the underwater light field

It is shown that where mu (s) is the average cosine of scattering, then for any set of photons that undergoes exactly n scatterings per photon, the average cosine after scattering is mu (0)mu (s)(n), where mu (0) is the average cosine of the photon flux before scattering. For a set of photons that has traversed distance d through a medium with scattering coefficient b, the average cosine is mu (0) exp[-bd(1 - mu (s))]. For water bodies in which loss of upward-scattered photons through the surface is small enough to be disregarded, the value of mu (c) (the average cosine of all the photons instantaneously present in the water column) for any given incoming flux of photons with average cosine mu (0) is determined entirely by the inherent optical properties of the water in accordance with mu (c)= mu (0)/[1 + (b/a)(1 - mu (s))], where a and b are the absorption and scattering coefficients.     

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.     

Empirical model functions to calculate hematocrit-dependent optical properties of human blood

The absorption coefficient, scattering coefficient, and effective scattering phase function of human red blood cells (RBCs) in saline solution were determined for eight different hematocrits (Hcts) between 0.84% and 42.1% in the wavelength range of 250-1100 nm using integrating sphere measurements and inverse Monte Carlo simulation. To allow for biological variability, averaged optical parameters were determined under flow conditions for ten different human blood samples. Based on this standard blood, empirical model functions are presented for the calculation of Hct-dependent optical properties for the RBCs. Changes in the optical properties when saline solution is replaced by blood plasma as the suspension medium were also investigated.     

T-matrix computations of light scattering by red blood cells

The electromagnetic far field, as well as the near field, originating from light interaction with a red blood cell (RBC)volume-equivalent spheroid, was analyzed by utilizing theT-matrix theory. This method is a powerful tool thatmakes it possible to study the influence of cell shape on the angulardistribution of scattered light. General observations were that thethree-dimensional shape, as well as the optical thickness apparent tothe incident field, affects the forward scattering. Thebackscattering was influenced by the shape of the surface facing theincident beam. Furthermore sphering as well as elongation of anoblate RBC into a volume-equivalent sphere or a prolate spheroid, respectively, was theoretically modeled to imitate physiologicalphenomena caused, e.g., by heat or the increased shear stress offlowing blood. Both sphering and elongation were shown to decreasethe intensity of the forward-directed scattering, thus yielding lowerg factors. The sphering made the scattering patternindependent of azimuthal scattering angle phi(s), whereas the elongation induced more apparent phi(s)-dependent patterns. The lightscattering by a RBC volume-equivalent spheroid was thus found to behighly influenced by the shape of the scattering object. Anear-field radius r(nf) was evaluated as thedistance to which the maximum intensity of the total near field haddecreased to 2.5 times that of the incident field. It was estimatedto 2-24.5 times the maximum radius of the scattering spheroid, corresponding to 12-69 mum. Because the near-field radiuswas shown to be larger than a simple estimation of the distance betweenthe RBC's in whole blood, the assumption of independent scattering, frequently employed in optical measurements on whole blood, seemsinappropriate. This also indicates that one cannot extrapolate theresults obtained from diluted blood to whole blood by multiplying witha simple concentration factor.     

Haar transform analysis of photon time-of-flight measurements for quantification of optical properties in scattering media

A method to independently quantify the absorption and the scattering properties of samples based on the analysis of the Haar transform (HT) of photon time-of-flight (TOF) distributions is described. A series of reflectance photon TOF measurements were acquired from absorbing/scattering milk samples of known composition (0 < mu(a) < 0.025 mm(-1); 100 < mu(s) < 250 mm(-1)). The HT of the profiles was calculated, and the regression based on the most parsimonious subset of wavelets was determined by the genetic algorithm (GA). In addition, the utility of computing the logarithm of the profiles or of the absolute value of the wavelet coefficients before the GA was studied. Results show that the absorption coefficient could be estimated with a coefficient of variation (C.V.) of 6.7% and an r2 of 0.99 by use of the log of selected wavelets of frequency less than 800 MHz. Scattering coefficients were estimated with a C.V. of 2.3% and an r2 of 0.99 with the log of wavelets of frequency less than 400 MHz. The above results suggest that a simplified instrument based on low-frequency switches could be developed to quantify the optical properties of highly scattering media.     

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.     

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.     

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.     

Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer

Gold nanoparticles with unique optical properties may be useful as biosensors in living whole cells. Using a simple and inexpensive technique, we recorded surface plasmon resonance (SPR) scattering images and SPR absorption spectra from both colloidal gold nanoparticles and from gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies after incubation in cell cultures with a nonmalignant epithelial cell line (HaCaT) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). Colloidal gold nanoparticles are found in dispersed and aggregated forms within the cell cytoplasm and provide anatomic labeling information, but their uptake is nonspecific for malignant cells. The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found to give a relatively sharper SPR absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells. These results suggest that SPR scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.     

Frequency-domain photon migration measurements of normal and malignant tissue optical properties in a human subject

A 1-GHz multifrequency, multiwavelength frequency-domain photon migration instrument is used to measure quantitatively the optical absorption (mu(a)) and effective optical scattering (mu(s) ?) of normal and malignant tissues in a human subject. Large ellipsoidal (~10-cm major axis, ~6-cm minor axes) subcutaneous malignant lesions were compared with adjacent normal sites in the abdomen and back. Absorption coefficients recorded at 674, 811, 849, and 956 nm were used to calculate tissue hemoglobin concentration (oxyhemoglobin, deoxyhemoglobin, and total), water concentration, hemoglobin oxygen saturation, and blood volume fraction in vivo. Our results show that the normal and the malignant tissues measured in the patient have clearly resolvable optical and physiological property differences that may be broadly useful in identifying and characterizing tumors.     

Scattering and absorption effects in the determination of glucose in whole blood by near-infrared spectroscopy

Optical properties of whole bovine blood are examined under conditions of different glucose loadings. A strong dependency is established between the scattering properties of the whole blood matrix and the concentration of glucose. This dependency is explained in terms of variations in the refractive index mismatch between the scattering bodies (predominately red blood cells) and the surrounding plasma. Measurements in the presence of a well-known glucose transport inhibitor indicate that variations in refractive index mismatch are related to the penetration of glucose into the red blood cells and demonstrate that increased scattering involves the uptake of glucose by red blood cells. Finally, multivariate calibration models are presented for the measurement of glucose in a whole blood matrix. These models are based on near-infrared spectral data collected from 80 different samples prepared from a single whole blood matrix. Calibration studies are performed over the combination, first-overtone, and short-wavelength spectral regions. The best calibration model is generated from combination region spectra, providing a standard error of prediction (SEP) of less than 1 mM over the concentration range of 3-30 mM. The model based on the first-overtone region is slightly degraded but still provides acceptable performance (SEP = 1.20 mM). The model based on the short-wavelength region is further degraded (SEP = 2.53 mM). To rationalize these results, an analysis of the selectivity of the calibration models is performed by computing the glucose net analyte signal. It is established that the models based on the combination and first-overtone regions are dominated by glucose absorption information, while the model computed from the short-wavelength region is based primarily on scattering information. This result provides evidence that absorption information is needed in order to obtain a glucose calibration model with acceptable performance.     

Broadband absorption spectroscopy in turbid media by combined frequency-domain and steady-state methods

A technique for measuring broadband near-infrared absorption spectra of turbid media that uses a combination of frequency-domain (FD) and steady-state (SS) reflectance methods is presented. Most of the wavelength coverage is provided by a white-light SS measurement, whereas the FD data are acquired at a few selected wavelengths. Coefficients of absorption (mu(a)) and reduced scattering (mu(s)') derived from the FD data are used to calibrate the intensity of the SS measurements and to estimate mu(s)' at all wavelengths in the spectral window of interest. After these steps are performed, one can determine mu(a) by comparing the SS reflectance values with the predictions of diffusion theory, wavelength by wavelength. Absorption spectra of a turbid phantom and of human breast tissue in vivo, derived with the combined SSFD technique, agree well with expected reference values. All measurements can be performed at a single source-detector separation distance, reducing the variations in sampling volume that exist in multidistance methods. The technique uses relatively inexpensive light sources and detectors and is easily implemented on an existing multiwavelength FD system.     

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.     

Three- and four-photon absorption of a multiphoton absorbing fluorescent probe

High-order multiphoton excitation processes are becoming a reality for fluorescence imaging and phototherapy treatment because they afford minimization of scattered light losses and a reduction of unwanted linear absorption in the living organism transparency window, making them less susceptible to photodamage, while improving the irradiation penetration depth and spatial resolution. We report the four-photon-excited fluorescence emission of (7-benzothiazol-2-yl-9,-didecylfluoren-2-yl)diphenylamine in hexane and its four-photon absorption cross section sigma4' = 8.1 x 10(-109) cm8 s3 photon(-3) for the transition S0 --> S1 when excited at 1600 nm with a tunable optical parametric generator (OPG) pumped by picosecond laser pulses. When pumped at 1200 nm, three-photon absorption was observed, corresponding to the same transition.