Performance of isotropic light dosimetry probes based on scattering bulbs in turbid media

In a previous paper the calibration of an isotropic light detector in clear media was described and validated. However, in most applications the detector is used to measure light distribution in turbid (scattering) media, that is, in tissues or tissue equivalent optical phantoms. Despite its small diameter (typically 0.8 mm), inserting the detector in a turbid medium may perturb the light distribution and change the fluence rate at the point of measurement. In the present paper we estimate the error in the fluence rate measured by a detector in turbid media after calibration in a clear medium (air), using an optical phantom and detector bulbs of different optical properties. The experimental results are compared with calculations using the diffusion approximation to the transport equation in a spherical geometry. From measurements in optical phantoms and the results of the calculations it appears that introduction of the detector into a water-based turbid medium with refractive index, absorption- and scattering coefficients different from those of the detector bulb may require corrections to the detector response of up to 10-15%, in order to obtain the true fluence rate in that medium. The diffusion model is used to explore the detector response in a number of tissues of interest in photodynamic therapy, using tissue optical properties from the literature. Based on these model calculations it is estimated that in real tissues the fluence rate measured by the detector is up to 3% below the true value.     

Balanced detection for low-noise precision polarimetric measurements of optically active, multiply scattering tissue phantoms

The use and advantages of balanced detection for making low-noise polarimetric measurements in turbid materials are demonstrated. The technique reduces the intensity noise originating from the laser and, in addition, makes possible a direct measurement of a component of the Stokes vector. When phase-locked detection is used with either amplitude or polarization modulation for polarimetric measurements in turbid media, one can obtain elements of the scattering matrix of very small magnitude. This methodology is used to measure optical activity and surviving linear polarization fractions in clear and turbid media containing glucose at physiologically relevant concentrations. The results are in agreement with Monte Carlo simulations of polarized light propagation in turbid media.     

Stokes polarimetry imaging of rat tail tissue in a turbid medium: degree of linear polarization image maps using incident linearly polarized light

Illumination with incident linearly polarized light on tissue and polarization state measurements of the remitted light provide a means by which various tissue structures can be differentiated. A rat tail is embedded within a turbid gelatin such that there is a variable depth of medium above it. By varying the incident polarization angle (IPA) of the illuminating linearly polarized light, the geometry, and the orientation angle of the tissue, a series of 2-D degree of linear polarization image maps are created using our Stokes polarimetry imaging technique. The image maps show locations of the polarization-sensitive structures in the rat tail, including soft tissue, intervertebral disks, and tendons. The observed morphologies in the image maps indicate locations where the depolarization of light differs according to the tissue type and underlying layers. The data indicate the importance of varying the IPA, and that tissue dichroism and birefringence affect the degree of linear polarization image maps. Diagnostic information regarding subsurface tissue structures is obtained.     

Review of polarization sensitive optical coherence tomography and Stokes vector determination

Polarization sensitive optical coherence tomography (PS-OCT) provides depth resolved measurements of the polarization state of light reflected from turbid media such as tissue. The theory and calculation of the Stokes vector of light reflected from turbid media is described and application of PS-OCT to contemporary biomedical imaging problems is given. Measurement of the depth resolved Stokes parameters allows determination of the degree of polarization and optical axis orientation in turbid media that can be modeled as a linear retarder. Effect of multiple scattering and speckle on the accuracy and noise of the computed Stokes parameters is discussed. Future directions for development of PS-OCT instrumentation for biological and medical applications is given.     

Iterative reconstruction method for light emitting sources based on the diffusion equation

Bioluminescent imaging (BLI) of luciferase-expressing cells in live small animals is a powerful technique for investigating tumor growth, metastasis, and specific biological molecular events. Three-dimensional imaging would greatly enhance applications in biomedicine since light emitting cell populations could be unambiguously associated with specific organs or tissues. Any imaging approach must account for the main optical properties of biological tissue because light emission from a distribution of sources at depth is strongly attenuated due to optical absorption and scattering in tissue. Our image reconstruction method for interior sources is based on the deblurring expectation maximization method and takes into account both of these effects. To determine the boundary of the object we use the standard iterative algorithm-maximum likelihood reconstruction method with an external source of diffuse light. Depth-dependent corrections were included in the reconstruction procedure to obtain a quantitative measure of light intensity by using the diffusion equation for light transport in semi-infinite turbid media with extrapolated boundary conditions.     

Angular measurements of light scattered by turbid chiral media using linear Stokes polarimeter

The effects of turbid chiral media on light polarization are studied in different directions around the scattering samples using a refined linear Stokes polarimeter, which simplifies the signal analysis, and allows for the detailed investigations of scattered light. Because no moving parts are involved in a measurement at a specific detection direction, the determination accuracy of polarization states is increased. The results show that light depolarization increases with both turbidity and detection angle for low and moderately turbid samples; however, the angular dependence decreases with increasing turbidity. When the turbidity is increased to approximately 100 cm(-1), the depolarization becomes higher in the forward than in the backward direction. Polarization sensitive Monte Carlo simulations are used to verify some experimental observations. The results also demonstrate that surviving linear polarization fraction and overall intensity are more sensitive to the increase of glucose concentration in backward than in the forward direction in highly turbid media, indicating that backward geometry may be preferable for potential glucose detection in a biomedical context. Comparison measurements with optically inactive glycerol suggest that the refractive index matching effect, and not the chiral nature of the solute, dominates the observed optical rotation engendered by glucose in highly turbid media.     

Propagation of polarized light in birefringent turbid media: a Monte Carlo study

A detailed study, based on a Monte Carlo algorithm, of polarized light propagation in birefringent turbid media is presented in this paper. Linear birefringence, which results from the fibrous structures, changes the single scattering matrix and alters the polarization states of photons propagating in biological tissues. Some Mueller matrix elements of light backscattered from birefringent anisotropic turbid media present unusual intensity patterns compared with those for nonbirefringent isotropic turbid media. This result is in good agreement with the analytic results based on the double-scattering model. Degree of polarization, Stokes parameters, and diffuse reflectance as functions of linearly birefringent parameters based on numerical results and theoretical analysis are discussed and compared in an effort to understand the essential physical processes of polarized light propagation in fibrous tissues.     

Drug quantification in turbid media by fluorescence imaging combined with light-absorption correction using white Monte Carlo simulations

Accurate quantification of photosensitizers is in many cases a critical issue in photodynamic therapy. As a noninvasive and sensitive tool, fluorescence imaging has attracted particular interest for quantification in pre-clinical research. However, due to the absorption of excitation and emission light by turbid media, such as biological tissue, the detected fluorescence signal does not have a simple and unique dependence on the fluorophore concentration for different tissues, but depends in a complex way on other parameters as well. For this reason, little has been done on drug quantification in vivo by the fluorescence imaging technique. In this paper we present a novel approach to compensate for the light absorption in homogeneous turbid media both for the excitation and emission light, utilizing time-resolved fluorescence white Monte Carlo simulations combined with the Beer-Lambert law. This method shows that the corrected fluorescence intensity is almost proportional to the absolute fluorophore concentration. The results on controllable tissue phantoms and murine tissues are presented and show good correlations between the evaluated fluorescence intensities after the light-absorption correction and absolute fluorophore concentrations. These results suggest that the technique potentially provides the means to quantify the fluorophore concentration from fluorescence images.     

Methodology for examining polarized light interactions with tissues and tissuelike media in the exact backscattering direction

The properties of polarized light emerging from turbid media in the exact backscattering direction are studied by modulating the incident light polarization state and isolating the synchronous signal with lock-in amplifier detection. The results are reported for polystyrene microsphere suspensions in distilled water, with and without glucose, and for both ex vivo and in vivo biological tissues. A new theoretical formulation based on Mueller calculus is developed to describe the observed behavior of the backscattered light in terms of two sample parameters: optical rotation and depolarization. This technique proved successful in modeling both phantom and tissue samples. Results showed the presence of a significant surviving polarization fraction in the backscattering direction even in extremely dense optical phantom media, an important finding that has not been observed at other detection angles. Substantial polarized light preservation in biological tissue samples is also demonstrated for this detection geometry. This illustrates the potential of using polarized light to investigate turbid biological materials in vivo in retroreflection geometry.     

Determination of optical properties in heterogeneous turbid media using a cylindrical diffusing fiber

For interstitial photodynamic therapy (PDT), cylindrical diffusing fibers (CDFs) are often used to deliver light. This study examines the feasibility and accuracy of using CDFs to characterize the absorption (μ(a)) and reduced scattering (μ'(s)) coefficients of heterogeneous turbid media. Measurements were performed in tissue-simulating phantoms with μ(a)?between 0.1 and 1?cm(-1)?and μ'(s) between 3 and 10?cm(-1)?with CDFs 2 to 4?cm in length. Optical properties were determined by fitting the measured light fluence rate profiles at a fixed distance from the CDF axis using a heterogeneous kernel model in which the cylindrical diffusing fiber is treated as a series of point sources. The resulting optical properties were compared with independent measurement using a point source method. In a homogenous medium, we are able to determine the absorption coefficient μ(a)?using a value of μ'(s) determined a priori (uniform fit) or μ'(s) obtained by fitting (variable fit) with standard (maximum) deviations of 6% (18%) and 18% (44%), respectively. However, the CDF method is found to be insensitive to variations in μ'(s), thus requiring a complementary method such as using a point source for determination of μ'(s). The error for determining μ(a)?decreases in very heterogeneous turbid media because of the local absorption extremes. The data acquisition time for obtaining the one-dimensional optical properties distribution is less than 8?s. This method can result in dramatically improved accuracy of light fluence rate calculation for CDFs for prostate PDT in vivo when the same model and geometry is used for forward calculations using the extrapolated tissue optical properties.     

Hybrid diffusion-P3 equation in N-layered turbid media: steady-state domain

This paper discusses light propagation in N-layered turbid media. The hybrid diffusion-P3 equation is solved for an N-layered finite or infinite turbid medium in the steady-state domain for one point source using the extrapolated boundary condition. The Fourier transform formalism is applied to derive the analytical solutions of the fluence rate in Fourier space. Two inverse Fourier transform methods are developed to calculate the fluence rate in real space. In addition, the solutions of the hybrid diffusion-P3 equation are compared to the solutions of the diffusion equation and the Monte Carlo simulation. For the case of small absorption coefficients, the solutions of the N-layered diffusion equation and hybrid diffusion-P3 equation are almost equivalent and are in agreement with the Monte Carlo simulation. For the case of large absorption coefficients, the model of the hybrid diffusion-P3 equation is more precise than that of the diffusion equation. In conclusion, the model of the hybrid diffusion-P3 equation can replace the diffusion equation for modeling light propagation in the N-layered turbid media for a wide range of absorption coefficients.     

Locating inhomogeneities in tissue by using the most probable diffuse path of light

Characterization of human tissue using near-IR (NIR) light is becoming increasingly popular. The light signal transmitted from the tissue contains information concerning inhomogeneities in tissue, such as size, position, and pathological states (benign or malignant). We discuss the most probable diffuse path (MPDP) related to frequency-domain diffuse photon density waves (DPDWs) propagating inside turbid media. We find that for a medium of finite size, the existence of boundaries between tissue and nonscattering media would have considerable impact on the path shape. It is also demonstrated that such paths can be used to obtain higher accuracy in localizing absorbers embedded in a homogeneous background. Based on the proposed MPDP, a new method for 3-D localization of heterogeneities in turbid media is proposed, which is validated by experiments using Intralipid and pork fat. The experiments are performed with an NIR breast cancer detection system designed and assembled in our lab, using 780-nm NIR light. In Intralipid, when the size of a single absorber is less than 1 cm, the localization error is about 2 mm. The results from pork fat are also acceptable.     

Time-reversed ultrasonically encoded optical focusing into tissue-mimicking media with thickness up to 70 mean free paths

In turbid media such as biological tissue, multiple scattering hinders direct light focusing at depths beyond one transport mean free path. As a solution to this problem, time-reversed ultrasonically encoded (TRUE) optical focusing is proposed based on ultrasonic encoding of diffused laser light and optical time reversal. In TRUE focusing, a laser beam of long coherence length illuminates a turbid medium, where the incident light undergoes multiple scattering and part of it gets ultrasonically encoded within the ultrasonic focal zone. A conjugated wavefront of the ultrasonically encoded light is then generated by a phase conjugate mirror outside the medium, which traces back the trajectories of the ultrasonically encoded diffused light and converges light to the ultrasonic focal zone. Here, we report the latest experimental improvement in TRUE optical focusing that increases its penetration in tissue-mimicking media from a thickness of 3.75 to 7.00 mm. We also demonstrate that the TRUE focus depends on the focal diameter of the ultrasonic transducer.     

Localization and quantification of fluorescent inclusions embedded in a turbid medium

A set-up, based on a CCD camera, to localize fluorescent inclusions in diffusing media was developed. This set-up allows one to acquire a huge dataset along two axes. This aspect is fundamental to performing a tomographic reconstruction in order to quantify the fluorescence amplitude in each voxel of the sample. Firstly, a simple analytical approach to recover the position of a single inclusion, embedded in a turbid medium, was developed. Then, we implemented a reconstruction algorithm to recover the position of one and two inclusions and to estimate their relative concentrations. Finally, we studied the dependence of reconstructed data on the number of injection points of excitation light and the number of detection points of fluorescence emission.     

Transillumination hyperspectral imaging for histopathological examination of excised tissue

Angular domain spectroscopic imaging (ADSI) is a novel technique for the detection and characterization of optical contrast in turbid media based on spectral characteristics. The imaging system employs a silicon micromachined angular filter array to reject scattered light traversing a specimen and an imaging spectrometer to capture and discriminate the largely remaining quasiballistic light based on spatial position and wavelength. The imaging modality results in hyperspectral shadowgrams containing two-dimensional (2D) spatial maps of spectral information. An ADSI system was constructed and its performance was evaluated in the near-infrared region on tissue-mimicking phantoms. Image-based spectral correlation analysis using transmission spectra and first order derivatives revealed that embedded optical targets could be resolved. The hyperspectral images obtained with ADSI were observed to depend on target concentration, target depth, and scattering level of the background medium. A similar analysis on a muscle and tumor sample dissected from a mouse resulted in spatially dependent optical transmission spectra that were distinct, which suggested that ADSI may find utility in classifying tissues in biomedical applications.     

Target detection and quantification using a hybrid hand-held diffuse optical tomography and photoacoustic tomography system

We present a photoacoustic tomography-guided diffuse optical tomography approach using a hand-held probe for detection and characterization of deeply-seated targets embedded in a turbid medium. Diffuse optical tomography guided by coregistered ultrasound, MRI, and x ray has demonstrated a great clinical potential to overcome lesion location uncertainty and to improve light quantification accuracy. However, due to the different contrast mechanisms, some lesions may not be detectable by a nonoptical modality but yet have high optical contrast. Photoacoustic tomography utilizes a short-pulsed laser beam to diffusively penetrate into tissue. Upon absorption of the light by the target, photoacoustic waves are generated and used to reconstruct, at ultrasound resolution, the optical absorption distribution that reveals optical contrast. However, the robustness of optical property quantification of targets by photoacoustic tomography is complicated because of the wide range of ultrasound transducer sensitivity, the orientation and shape of the targets relative to the ultrasound array, and the uniformity of the laser beam. We show in this paper that the relative optical absorption map provided by photoacoustic tomography can potentially guide the diffuse optical tomography to accurately reconstruct target absorption maps.     

Measuring Tissue Properties and Monitoring Therapeutic Responses Using Acousto-Optic Imaging

Acousto-optic imaging is a hybrid imaging technique that exploits the interaction between light and sound to image optical contrast at depth in optically turbid media with the high spatial resolution of ultrasound. Quantitative measurement of optical properties using this technique is confounded by multiple parameters that influence the detected acousto-optic signal. In this article, we describe the origin of the acousto-optic response and review techniques that have been proposed to relate this response to the optical properties of turbid media. We present an overview of two acousto-optic sensing approaches. In the first, we demonstrate that the local transport mean free path within turbid media can be obtained by varying the pressure of the ultrasound field and processing the resulting acousto-optic signals. In the second, we demonstrate that the acousto-optic response elicited by a high-intensity ultrasound field during thermal therapy can be used to monitor the onset of lesion formation, ascertain lesion volume, and provide real-time control of exposure duration.     

Optical imaging of turbid media using independent component analysis: theory and simulation

A new imaging approach for 3-D localization and characterization of objects in a turbid medium using independent component analysis (ICA) from information theory is developed and demonstrated using simulated data. This approach uses a multisource and multidetector signal acquisition scheme. ICA of the perturbations in the spatial intensity distribution measured on the medium boundary sorts out the embedded objects. The locations and optical characteristics of the embedded objects are obtained from a Green's function analysis based on any appropriate model for light propagation in the background medium. This approach is shown to locate and characterize absorptive and scattering inhomogeneities within highly scattering medium to a high degree of accuracy. In particular, we show this approach can discriminate between absorptive and scattering inhomogeneities, and can locate and characterize complex inhomogeneities, which are both absorptive and scattering. The influence of noise and uncertainty in background absorption or scattering on the performance of this approach is investigated.     

Simulation study of second-harmonic microscopic imaging signals through tissue-like turbid media

We establish, for the first time, a simulation model for dealing with the second-harmonic signals under a microscope through a tissue-like turbid medium, based on the Monte Carlo method. With this model, the angle-resolved distribution and the signal level eta of second-harmonic light through a slab of the turbid medium are demonstrated and the effects of the thickness (d) of the turbid medium, the numerical aperture (NA) of the objective as well as the size (rho) of the scatterers forming the turbid medium are explored. Simulation results reveal that the use of a small objective NA results in a narrow angle distribution but strong second-harmonic signals. A turbid medium consisting of large scattering particles has a strong influence on the angle distribution and the signal level eta, which results in a low penetration limit for second-harmonic signals made up of ballistic photons. It is approximately 30 microm in our situation.     

Two-dimensional phased arrays of sources and detectors for depth discrimination in diffuse optical imaging

We present a multisource, multidetector phased-array approach to diffuse optical imaging that is based on postprocessing continuous-wave data. We previously showed that this approach enhances the spatial resolution of diffuse optical imaging. We now demonstrate the depth discrimination capabilities of this approach and its potential to perform tomographic sectioning of turbid media. The depth discrimination results from the dependence of the sensitivity function on the depth coordinate z. To demonstrate the potential of this approach, we perform an experimental study of a turbid medium containing cylindrical inhomogeneities that are placed 2.0, 3.0, and 4.0 cm from a seven-element, 2-D source array. A single detector element is placed at a distance of 6.0 cm from the source array, and the measurement is repeated after switching the positions of the detector and the source array to simulate the case where both sources and detectors consist of a 2-D array of elements. We find that the proposed phased-array method is able to separate cylinders at different depths, thus showing cross-sectioning capabilities.