Determination of scattering volume fraction and particle size distribution in the superficial layer of a turbid medium by using diffuse reflectance spectroscopy

We have investigated the possibility of determining changes in the volume fraction of microstructure scatterers in the superficial tissue layers by using diffuse reflectance spectroscopy. To that extent we have built a two-layer optical phantom by using microparticles with various sizes in order to simulate the scattering properties of tissue microstructures. Reflectance spectral measurements were performed on a number of optical phantoms having different volume fractions of various microparticle sizes. An analytical model was developed using light-transport theory and fractal modeling approaches and was then fitted to the measured reflectance to calculate the volume fractions of the microparticles in phantoms. The results showed that we could measure changes in both the total volume fraction of the microparticles and in the overall size distribution of the microparticles with good accuracy (>80%). These results suggest the potential of using this method for measuring the volume fraction changes of tissue microstructure scatterers and applications in the detection of cancerous related morphological and structural changes.     

Raman signal enhancement in deep spectroscopy of turbid media

A new, passive method for enhancing spontaneous Raman signals for the spectroscopic investigation of turbid media is presented. The main areas to benefit are transmission Raman and spatially offset Raman spectroscopy approaches for deep probing of turbid media. The enhancement, which is typically several fold, is achieved using a multilayer dielectric optical element, such as a bandpass filter, placed within the laser beam over the sample. This element prevents loss of the photons that re-emerge from the medium at the critical point where the laser beam enters the sample, the point where major photon loss occurs. This leads to a substantial increase of the coupling of laser radiation into the sample and consequently an enhanced laser photon-medium interaction process. The method utilizes the angular dependence of dielectric optical elements on impacting photon direction with its transmission spectral profile shifting to the blue with increase in the deviation of photons away from normal incidence. This feature enables it to act as a unidirectional mirror passing a semi-collimated laser beam through unhindered from one side, and at the other side, reflecting photons emerging from the sample at random directions back into it with no restrictions to the detected Raman signal. With substantial restrictions to the spectral range, the concept can also be applied to conventional backscattering Raman spectroscopy. The use of additional reflective elements around the sample to enhance the Raman signal further is also discussed. The increased signal strength yields higher signal quality, a feature important in many applications. Potential uses include sensitive noninvasive disease diagnosis in vivo, security screening, and quality control of pharmaceutical products. The concept is also applicable in an analogous manner to other types of analytical methods such as fluorescence or near-infrared (NIR) absorption spectroscopy of turbid media or it can be used to enhance the effectiveness of the coupling of laser radiation into tissue in applications such as photodynamic therapy for cancer treatment.     

Inverse spatially offset Raman spectroscopy for deep noninvasive probing of turbid media

A new type of highly sensitive spatially offset Raman spectroscopy (SORS) developed for deep noninvasive probing of stratified turbid media is described. The technique, termed inverse SORS, permits much greater depths to be interrogated than those accessible with the conventional SORS approach. This is achieved by enhancing the sensitivity of the technique through the elimination of spectral distortions inherent to the conventional SORS methodology. The method also permits the use of higher laser powers in applications where intensity limits exist, such as when probing human tissue in vivo. In addition, the new approach possesses a much higher degree of flexibility, enabling on-the-spot tailoring of experimental conditions such as the magnitude and number of spatial offsets to individual samples. The scheme uses a reverse SORS geometry whereby Raman light is collected through fibers at the center of the probe and laser radiation is delivered to the sample through a beam in the shape of a ring. The method is demonstrated on a layered powder sample and several practical examples of its uses, presented for the first time, are also given. Potential applications include disease diagnosis, noninvasive probing of pharmaceutical products and chemicals through packaging, probing of polymers, biofilms or paints, and homeland security screening.     

Technique for enhancing signal in conventional backscattering fluorescence and Raman spectroscopy of turbid media

We demonstrate experimentally, for the first time, the feasibility of passively enhancing fluorescence and Raman signals from diffusely scattering media in a conventional backscattering collection geometry. The method employs transmission of the collimated excitation laser beam through a "unidirectional" dielectric mirror placed directly in front of the sample. This permits laser light that escapes from the sample surface to be reflected back into the sample where it can be more usefully employed in generating Raman and fluorescence signals. This leads to improved Raman signal, higher signal-to-noise ratio, and shorter acquisition times. Feasibility studies performed on standard pharmaceutical tablets and on sheets of Teflon, using a single enhancing element, demonstrate signal enhancement factors of 6 (fluorescence) and 3 (Raman). Potential applications of this simple device include improving quality control of pharmaceutical products, disease diagnosis of biological tissue, forensics, and security screening.     

Prediction of sublayer depth in turbid media using spatially offset Raman spectroscopy

We demonstrate experimentally the feasibility of monitoring the depth of optically thick layers within turbid media using spatially offset Raman spectroscopy (SORS) in combination with multivariate analysis. The method uses the deep penetration capability of SORS to characterize significantly thicker (by at least a factor of 2) layers than possible with conventional Raman spectroscopy. Typical relative accuracies were between 5 and 10%. The incorporation of depth information into a SORS experiment as an additional dimension allows pure spectra of each individual layer to be resolved using three-dimensional multivariate techniques (parallel factor analysis, PARAFAC) to accuracies comparable with the results of a two-dimensional analysis.     

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.     

Measurement of the local optical properties of turbid media by differential path-length spectroscopy

We report on the development of an optical-fiber-based diagnostic tool with which to determine the local optical properties of a turbid medium. By using a single fiber in contact with the medium to deliver and detect white light, we have optimized the probability of detection of photons scattered from small depths. The contribution of scattered light from greater depths to the signal is measured and subtracted with an additional fiber, i.e., a collection fiber, to yield a differential backscatter signal. Phantoms demonstrate that, when photons have large mean free paths compared with the fiber diameter, single scattering dominates the differential backscatter signal. When photons have small mean free paths compared with the fiber diameter, the apparent path length of the photons that contribute to the differential backscatter signal becomes approximately equal to 4/5 of the fiber diameter. This effect is nearly independent of the optical properties of the sample under investigation.     

Multiple path analysis of reflectance from turbid media

A novel method to calculate the reflectance of light from a turbid medium is presented. The method takes an approach similar to that of the Beer-Lambert law, where the intensity of light is attenuated by an exponential factor involving the path length and the absorption coefficient. Due to scatter, however, there are many path lengths; in the present method all possible path lengths are weighted by their probabilities and summed over. A path length probability density is derived by considering a photon random walk through the medium. The result is a simple expression for the reflectance based on the physical properties of the medium.     

Assessment of the upper particle size limit for quantitative analysis of aerosols using laser-induced breakdown spectroscopy

The laser-induced plasma vaporization of individual silica microspheres in an aerosolized air stream was investigated. The upper size limit for complete particle vaporization corresponds to a silica particle diameter of 2.1 microm for a laser pulse energy of 320 mJ, as determined by the deviation from a linear mass response of the silicon atomic emission signal. Comparison of the measured silica particle sampling rates and those predicted based on Poisson sampling statistics and the overall laser-induced plasma volume suggests that the primary mechanism of particle vaporization is related to direct plasma-particle interactions as opposed to a laser beam-particle interaction. Finally, temporal and spatial plasma evolution is discussed in concert with factors that may influence the vaporization dynamics of individual aerosol particles, such as thermophoretic forces and vapor expulsion.     

Three-dimensional imaging of objects embedded in turbid media with fluorescence and Raman spectroscopy

We present a single-ended technique for three-dimensional imaging of objects embedded in a turbid medium by the use of time-resolved fluorescence emission or Raman scattering. The technique uses the earliest arriving photons, which we show are not sensitive to the relatively long fluorescence lifetime, and thus can be used to extract the desired spatial information accurately, even at a distance equivalent to 100 mean free paths. The results also demonstrate the feasibility and the potential of one's combining time-resolved optical tomography with fluorescence or Raman spectroscopy to localize and identify the embedded objects. This technique may be valuable for the diagnosis of disease in highly scattering human tissue because it can provide spatial and biochemical information about the composition of embedded lesions.     

Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption

The fluorescence from a turbid medium such as biologic tissue contains information about scattering and absorption, as well as the intrinsic fluorescence, i.e., the fluorescence from an optically thin sample of pure fluorophores. The interplay of scattering and absorption can result in severe distortion of the intrinsic spectral features. These distortions can be removed by use of a photon-migration-based picture and information from simultaneously acquired fluorescence and reflectance spectra. We present experimental evidence demonstrating the validity of such an approach for extracting the intrinsic fluorescence for a wide range of scatterer and absorber concentrations in tissue models, ex vivo and in vivo tissues. We show that variations in line shape and intensity in intrinsic tissue fluorescence are significantly reduced compared with the corresponding measured fluorescence.     

Deep subsurface Raman spectroscopy of turbid media by a defocused collection system

A simple procedure for the recovery of deep subsurface Raman spectra in stratified turbid samples by defocusing a conventional Raman instrument is presented. The method is based on effects present with spatially offset Raman spectroscopy (SORS) and, although not as efficient as the standard SORS approach, it permits a simple way of recovering subsurface Raman spectra from less challenging samples. Demonstration of the effect is performed using a standard SORS device and a commercial Raman instrument on the noninvasive measurement of paracetamol tablets held within a nontransparent white plastic bottle.     

Quantitative microspectrophotometry in turbid media

We present a new technique to determine the scattering coefficient, the absorption coefficient, and the anisotropy factor in turbid media on a microscopic level. To this end, a microspectrophotometer was used to obtain transmission measurements at different solid angles. To extract the optical properties from phantom materials (liquid and solid) and biological tissue (bovine liver) an inverse Monte Carlo algorithm was used. The results obtained with the new microspectrophotometric technique agreed within one standard deviation with the values from Mie theory and within less than 10% with the data derived from conventional spectroscopic measurements. The results suggest that this new method is a valid tool to determine the optical properties of turbid media on a microscopic level.     

Controlling the optical path length in turbid media using differential path-length spectroscopy: fiber diameter dependence

We have characterized the path length for the differential path-length spectroscopy (DPS) fiber optic geometry for a wide range of optical properties and for fiber diameters ranging from 200 microm to 1000 microm. Phantom measurements show that the path length is nearly constant for scattering coefficients in the range 5 mm(-1)< micros <50 mm(-1) for all fiber diameters and that the path length is proportional to the fiber diameter. The path length decreases with increasing absorption for all fiber diameters, and this effect is more pronounced for larger fiber diameters. An empirical model is formulated that relates the DPS path length to total absorption for all fiber diameters simultaneously.     

Limited possibility for quantifying mean particle size by logarithmic light-scattering spectroscopy

Recent studies have shown that the slope of logarithmic scattering spectroscopy of a turbid medium is related to the sizes of the scattering particles within the turbid medium. Mie theory can be used to generate a logarithmic plot of the reduced-scattering coefficient versus wavelength. According to Nilsson et al. [Appl. Opt. 37, 1256 (1998)], the slope value of a linear fit of the logarithmic scattering spectroscopy between 600 and 1050 nm can be used for direct determination of particle size. We performed similar calculations using the Rayleigh-Gans approximation and obtained an analogous overall shape with additional sinusoidal features. Our calculations indicate a possible relationship between the slope and the particle size when the size is used to calculate the slope, namely, in the forward calculation. However, because of the sinusoidal pattern, the inverse calculation to obtain the particle size from the slope may be applied only for particles with a radius of <0.13 microm in combination with 650-1050-nm light. Caution should be exercised when inverse calculation is performed to determine the scattering particle sizes in the range of radii >0.13 microm, with the slope of logarithmic scattering spectroscopy within 650-1050 nm.     

Particle size analysis of turbid media with a single optical fiber in contact with the medium to deliver and detect white light

Wavelength-dependent elastic-scattering spectra of highly scattering media measured with only a single fiber for both light delivery and collection are presented. White light is used as a source, and oscillations of the detected light intensities are seen as a function of wavelength. The maximum and minimum of the oscillations can be used to determine scatterer size for monodisperse distributions of spheres when the refractive indices are known. In addition, several properties of the probe relevant to tissue diagnosis are examined and discussed including the effects of absorption, a broad distribution of scatterers, and the depth probed.     

Static and dynamic properties of highly turbid media determined by spatially resolved diffusive-wave spectroscopy

We show that spatially resolved backscattering can be used for simultaneous measurements of static and dynamic properties of highly turbid media. The spatial variation of the backscattered intensity gives access to the transport men free path. The decay of the temporal intensity-intensity correlation function depends on the point of observation. This property can be used to probe complex dynamics with several time scales. The implementation of the method and the data analysis are tested on concentrated suspensions of polystyrene spheres.     

Dual-spatial integration for longitudinal localization of inclusions in turbid media

We introduce a technique called dual-spatial integration (DSI) that is used to isolate and enhance inclusions that differ only by their longitudinal placement within a scattering medium. DSI uses three different source-detector configurations to section a scattering medium into three longitudinal zones. This sectioning permits the extraction of structures close to surfaces and the enhancement of those structures located in the central part of the medium. Both the simulation and the experimental results indicate that DSI has potential interest for applications in biomedical imaging such as optical mammography.     

Improved inversion procedure for particle size distribution determination by photon correlation spectroscopy

We propose a minimum variation of solution method to determine the optimal regularization parameter for singular value decomposition for obtaining the initial distribution for a Chahine iterative algorithm used to determine the particle size distribution from photon correlation spectroscopy data. We impose a nonnegativity constraint to make the initial distribution more realistic. The minimum variation of solution is a single constraint method and we show that a better regularization parameter may be obtained by increasing the discrimination between adjacent values. We developed the S-R curve method as a means of determining the modest iterative solution from the Chahine algorithm. The S-R curve method requires a smoothing operator. We have used simulated data to verify our new method and applied it to real data. Both simulated and experimental data show that the method works well and that the first derivative smoothing operator in the S-R curve gives the best results.     

Influence of size parameter and refractive index of the scatterer on polarization-gated optical imaging through turbid media

The influence of incident polarized light, refractive index, and size parameter of the scatterer on achievable resolution and contrast (image quality) of polarization-gated transillumination imaging in turbid media is reported here. Differential polarization detection led to significant improvement of image quality of an object embedded in a medium of small-sized scatterers (diameter Dor=lambda,g>or=0.7), the improvement in image quality was less pronounced using either linear or circular polarization gating when the refractive index of the scatterer was high (ns=1.59), but for a lower value of refractive index (ns=1.37), image quality improved with the differential circular polarization gating. We offer a plausible explanation for these observations.