Tissue polarimetry is an emerging area of technology development in tissue optics that is expected to lead to important research applications in biology and medicine. Traditional polarimetry is a mature field, but its application to thick tissues has been hindered by technical hurdles associated with multiple light scattering.

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.

The polarization properties of light scattered in a lateral direction from turbid media were studied. Polarization modulation and synchronous detection were used to measure, and Mueller calculus to model and derive, the degrees of surviving linear and circular polarization and the optical rotation induced by turbid samples. Polystyrene microspheres were used as scatterers in water solutions containing dissolved chiral, racemic, and achiral molecules. The preservation of circular polarization was found to exceed the linear polarization preservation for all samples examined. The optical rotation induced increased with the chiral molecule concentration only, whereas both linear and circular polarizations increased with an increase in the concentrations of chiral, racemic, and achiral molecules. This latter effect was shown to stem solely from the refractive index matching mechanism induced by the solute molecules, independent of their chiral nature.

We report the depolarization of light scattered by a variety of birefringent and nonbirefringent tissues. We used Stokes polarimetry to investigate how scatterer structures in each tissue contribute to the depolarization of linearly versus circularly polarized light propagating through that tissue. Experiments were performed on porcine blood, fat, tendon, artery, and myocardium. The results indicate that the two incident polarization states are depolarized differently depending on the structure of the sample. As seen in sphere suspensions, for tissues containing dilute Mie scatterers, circularly polarized light is maintained preferentially over linearly polarized light. For more dense tissues, however, the reverse is true. The results illustrate situations where polarized light will provide an improvement over unpolarized light imaging, information that is crucial to optimizing existing polarimetric imaging techniques.

The degree of polarization (DOP) of laser-speckle fields, where the speckles were generated by a polarized laser beam incident upon two kinds of samples: ground glass and wax, was investigated within a single coherence area as well as over multiple coherence areas. For the surface-scattering ground glass, the incident polarization state was preserved in the speckle field, and hence the DOP remained at unity regardless of the area of detection. For the volumescattering wax, the polarization states varied with positions in the field, and consequently the DOP depended on the area of detection: the DOP decreased with an increasing area of detection, and only when the area was much smaller than the coherence area would the DOP approach unity. A numerical simulation explained the experimental observation. These results are important for the understanding of polarization phenomena in turbid media such as biological tissue.

The scattering of polarized light from a two layer scattering medium is investigated using Monte Carlo simulations. First order and normalized second order moments are used to analyze the spatial properties of the emerging light in different polarization states. Linearly and circularly polarized illumination is used to probe different depths. Absorption and layer thickness are varied and it is demonstrated that the determination of these values is aided by the inclusion of polarization information. The lateral and depth localization of light by polarization subtraction is also quantified. Potential applications of these techniques are burn depth and melanoma thickness measurements.

Over the last two decades polarimetry has been investigated as a noninvasive alternative for glucose monitoring in support of diabetic patients. In particular, the anterior chamber of the eye containing the fluid known as the aqueous humor has been confirmed to be the optimal sensing site for polarimetric glucose measurements due to its reasonable pathlength (1 cm), low scatter, and minimal depolarization index. In essence, the eye can be thought of as an optical window into the body. In this paper, we will first introduce the key challenges that must be overcome to make the use of polarized light in the eye a viable method for noninvasive glucose monitoring, summarize our work toward this endeavor, and then report on our latest research, namely, the effect of temperature, pH , and corneal birefringence on our polarimetric glucose monitoring system.

Linearly polarized light that illuminates skin is backscattered by superficial layers and rapidly depolarized by birefringent collagen fibers. It is possible to distinguish such superficially backscattered light from the total diffusely reflected light that is dominated by light penetrating deeply into the dermis. The method involves acquisition of two images through an analyzing linear polarizer in front of the camera, one image (I par) acquired with the analyzer oriented parallel to the polarization of illumination and one image (I per) acquired with the analyzer oriented perpendicular to the illumination. An image based on the polarization ratio, Pol5(I par-I per)/(I par+I per), is created. This paper compares normal light images, represented by I per , and Pol images of various skin pathologies in a pilot clinical study using incoherent visible-spectrum light. Images include pigmented skin sites (freckle, tattoo, pigmented nevi) and unpigmented skin sites [nonpigmented intradermal nevus, neurofibroma, actinic keratosis, malignant basal cell carcinoma, squamous cell carcinoma, vascular abnormality (venous lake), burn scar]. Images of a shadow cast from a razor blade onto a forearm skin site illustrate the behavior of Pol values near the shadow edge. Near the shadow edge, Pol approximately doubles in value because no I per photons are superficially scattered into the shadow-edge pixels by the shadow region while I par photons are directly backscattered from the superficial layer of these pixels. This result suggests that the point spread function in skin for cross-talk between Pol pixels has a half-width-half-max of about 390 µm.

The high fatality rate associated with the late detection of skin cancer makes early detection crucial in preventing death. The current method for determining if a skin lesion is suspect to cancer is initially based on the patient’s and physician’s subjective observation of the skin lesion. Physicians use a set of parameters called the ABCD (asymmetry, border, color, diameter) rule to help facilitate diagnosis of potential cancerous lesions. Lesions that are suspicious then require a biopsy, which is a painful, invasive, and a time-consuming procedure. In an attempt to reduce the aforementioned undesirable elements currently associated with skin cancer diagnosis, a novel optical polarization-imaging system is described that has the potential to noninvasively detect cancerous lesions. The described system generates the full 16-element Mueller matrix in less than 70 s. The operation of the system was tested in transmission, specular reflection, and diffuse reflectance modes, using known samples, such as a horizontal linear polarizer, a mirror, and a diffuser plate. In addition, it was also used to image a benign lesion on a human subject. The results of the known samples are in good agreement with their theoretical values with an average accuracy of 97.96% and a standard deviation of 0.0084, using 16 polarization images. The system accuracy was further increased to 99.44% with a standard deviation of 0.005, when 36 images were used to generate the Mueller matrix.

Two-dimensional depth-resolved Jones-matrix images of scattering biological tissues were measured with novel double-source double-detector polarization-sensitive optical coherence tomography (OCT). The Jones matrix can be determined in a single scan with this OCT system. The experimental results show that this system can be effectively applied to the measurement of soft tissues, which are less stable than hard tissues. Polarization parameters such as diattenuation, birefringence, and orientation of the fast axis can be extracted through decomposition of the measured Jones matrix. The Jones matrix of thermally treated porcine tendon showed a reduction of birefringence from thermal damage. The Jones matrices of porcine skin and bovine cartilage also revealed that the density and orientation of the collagen fibers in porcine skin and bovine cartilage are not distributed as uniformly as in porcine tendon. Birefringence is sensitive to changes in tissue because it is based on phase contrast.

Polarization sensitive optical coherence tomography (PSOCT) 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.

In this paper, image enhancement and reconstruction through a turbid medium by utilizing polarization gating and mathematical image reconstruction methods in a microscopic imaging system are investigated. A Monte Carlo simulation model based on Mie theory and the concept of the effective point spread function (EPSF) is adopted to study image formation under a transmission-mode microscope. The results show that polarization gating methods, and particularly the differential polarization gating method, can be efficient in suppressing highly scattered light, which leads to a significant enhancement of image quality. An image reconstruction method based on the concept of the EPSF is demonstrated to be efficient in further improving image quality.

An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 µm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods.

We present the design and construction of a fiber optic probe for elastic light scattering spectroscopy in vivo with polarized excitation and polarization sensitive detection. The performance of the fiber probe is evaluated using a suspension of polystyrene spheres placed atop a diffusely scattering substrate, and it demonstrates that the size-dependent characteristics of the scatterers can be extracted in the presence of a highly diffusely scattering background using a linear combination of forward and backward Mie scattering components of the scatterers. Subsequently, Mie theory calculations are performed over a broad range of diagnostically relevant parameters of nuclei— mean diameter, size distribution, and relative refractive index—to understand how the polarized reflectance measurements with the fiber probe can be used to extract morphological information about epithelial tissue. Finally, the feasibility of in vivo measurements with the fiber optic based polarization sensitive light scattering spectroscopy is demonstrated.

Normal and malignant human cervical cancer cells were imaged in vivo with confocal, phase contrast, and brightfield microscopies. Results were compared between cells in growth medium before and after addition of acetic acid, hypertonic saline solution, toluidine blue, and Lugol’s iodine. The exogenous agents changed the backscattering characteristics of the cells when measured with confocal microscopy at 808 nm. A tendency toward higher scattering was observed in treated cells. Acetic acid and toluidine blue increased the brightness of the nucleus with respect to the cytoplasm in normal and cancer cells. Hypertonic saline solution made the cytoplasm brighter than the nucleus in both types of cells. The results indicate that simple chemicals can be used to enhance confocal microscopy’s ability to differentiate intracellular components, such as nuclear size and shape. This can further confocal microscopy’s ability to assess disease in cells and tissues.

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. In this paper, we describe the application of the oxygen sensing indicator, ruthenium dibipyridine 4-(1"-pyrenyl)-2,2'-bipyridine chloride [Ru(bpy-pyr)(bpy)2], for molecular oxygen measurement in J774 murine macrophages. Ru(bpy-pyr)(bpy)2 exhibits strong visible absorption, efficient fluorescence, long excited state lifetime, large Stokes shift, and high photoand chemical stability. The fluorescence of Ru(bpy-pyr)(bpy)2 is efficiently quenched by molecular oxygen. It is 13 fold higher in a nitrogenated solution than in an oxygenated one. The dye passively permeates into cells and maintains its oxygen sensitivity for at least 5 h when the cells are stored in a phosphate buffered saline solution at pH 7.4. The oxygen sensitivity, photostability, and chemical stability of the indicator and the effect of hypoxia and hyperoxia on the intracellular oxygen level in single macrophages are discussed.

The plasma membrane of Chinese hamster ovary cells was made permeable using the focused beam of an argon ion laser (488 nm) and phenol red as a light absorbing dye. Small circular dark spots on the cell surface appeared immediately after laser irradiation and disappeared within about 5 min. They were related to transient changes in membrane properties, which could be visualized using the fluorescent marker laurdan, and were probably due to a local increase in temperature. According to a colony forming assay, cell viability was maintained by using light doses up to 2.5 MJ/cm2 applied for 1 s. In addition to measurements of the efflux of the cytoplasmic marker calcein, cell transfection using a green fluorescent protein (GFP) coding plasmid was studied: brightly fluorescent GFP with an emission maximum around 510 nm was observed within part of the cells after 24 h. The transfection rates after laser irradiation were around 30% for younger subcultures and less than 10% for aging cells. This may be due to age dependent changes in the phase transition of membrane lipids from gel phase to liquid crystalline phase. High transfection rates, visual control and universality towards various cell lines are possibly the main advantages of laser-assisted optoporation in comparison with presently existing methods of cell transfection.

Synchrotron radiation-based Fourier transform infrared spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR) spectra, hence chemical information, with high signal to noise at spatial resolutions as fine as 3–10 µm. Mid-IR photons are too low in energy (0.05–0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, ‘‘Does the intense synchrotron beam induce any cytotoxic effects in living cells?’’ In this work, we present the results from a series of standard biological assays to evaluate any short- and/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using a 3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. All control, 5, 10, and 20 min SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable biochemical changes between control and exposed cells.

A new method for the study of drug–cell interaction is suggested. It is based on optical monitoring of cell response to drug impact. To detect this response, a high sensitive photothermal imaging technique has been developed. This technique is based on irradiation of the cells with a focused short pulse double-frequency pump laser (532 nm, 8 ns) and monitoring the laser-induced local thermal effects. This is realized with a second collinear dye laser pulse (630 nm, 6 ns) that probes the heated cell absorbing zones. The temperaturedependent variations of the refractive index in the absorbing zones in cross section of probe beam are recorded with phase-contrast technique. The photothermal (PT) image represents a two-dimensional depth-integrated temperature distribution in the irradiated volume and correlates with the conventional optical absorption images. It was experimentally shown that the introduction of NaN3 or anti-tumor drug in the cell suspension led to a change in the structure of PT images of human lymphocytes and human lympholeukemia cells, respectively. It was hypothesized that the observed effects could be associated with the change of redox state of respiratory chain component.

Raman scattering from membranes, cells, and tissues must all be anisotropic, because the molecular orientations in these biological systems are anisotropic. How can such observed Raman scattering anisotropy be related with a biologically relevant molecular arrangement? This question is the subject of this paper. A general method of addressing this question will be given, with three examples illustrating the use of the method: (1) carotenoid arrangement in the eyespot of Chlamydomonas, (2) orientation of the tryptophan side chain in the coat subunit of a filamentous virus, and (3) polypeptide orientation in fowl feather barb.

Background and objectives: Femtosecond pulses can generate high precision subsurface photodisruption in transparent tissues, such as the cornea. We used femtosecond laser technology to demonstrate early proof of concept for high precision subsurface photodisruption in the translucent sclera. This technique may ultimately enable novel surgical procedures for the treatment of glaucoma and/or presbyopia. Study design/materials and methods: Microjoule femtosecond pulses from two different sources, 1060 and 775 nm, were used to make subsurface incisions in human sclera in vitro. Scleral tissue was dehydrated to improve translucency at these wavelengths. The beam was focused to a 1.5 (775 nm) or 5 µm spot size (1060 nm) and scanned below the tissue surface at various depths to produce four incision patterns. Results: Photodisruption on the backsurface of the sclera was achieved without damage to overlying tissue. Several types of intrascleral incisions were made, including transcleral channels and grooves for scleral implants. Conclusions: High precision, subsurface scleral photodisruption can be achieved in vitro for a variety of intrascleral incisions. Further studies are required to determine if this technique is applicable in vivo for actual surgical applications.

Purpose: To study the retina in normal subjects with a highresolution imaging system using adaptive optics for wave front aberration correction. Methods: We used a low-cost 37-element micromachined membrane deformable mirror (MMDM) with a continuous membrane as the reflective surface. A Hartmann–Shack wave front sensor with cooled charge coupled device camera was used to measure the wave front aberration. Zernike polynomials were used to describe the wave front shape. We developed a mirror control system to compensate for wave aberrations. We tested this instrument in normal subjects. Results: We were able to image the retina in monochromatic laser light and document the increase in resolution. While it is hard to estimate the exact size of the smallest structures in the image, we were able to subjectively grade the image quality. The system is able to compensate for higher order aberrations present in the human eye. Conclusion: The capabilities of correcting ocular aberrations are limited by the number of adjustable elements in the mirror and the deflection range of the surface. The advantage of the MMDM system is its low cost when compared with other adaptive optics solutions such as piezodriven mirrors and spatial light modulators. This technique may allow for improved resolution for clinical fundus photography.

We present what is to our knowledge the first in vivo tomograms of human retina obtained by Fourier domain optical coherence tomography. We would like to show that this technique might be as powerful as other optical coherence tomography techniques in the ophthalmologic imaging field. The method, experimental setup, data processing, and images are discussed.

Tracking a bolus of contrast agent traveling through the cerebral vasculature provides a measure of the blood flow velocity in the respective cerebral tissue. This principle has been the basis for the first approaches in functional magnetic resonance (MR) imaging and is of great value for investigating patients with vascular disease, especially stroke. While bolus measurements are a standard procedure in MR imaging, optical bolus tracking is as yet not established. Here we study optical absorption changes induced by a bolus of the dye indocyanine-green with near infrared spectroscopy in healthy volunteers. The aim is to assess the latency and shape of the change in absorption. Since application in the adult human critically depends on differentiation between extra- and intracerebral vascular compartments we focus on an approach for such a separation. To do this frequency-domain and multidistance measurements are analyzed by a Monte Carlo based model for photon transport in tissue. Based on measurements of both the photon’s mean time of flight (phase) and the intensity, our results allow differentiation between an upper (skin and skull) and a lower layer (brain). The bolus in the deeper tissue layers has a peak of about 10 s width, while the change in absorption in the upper layers shows a much longer recovery time. This is in qualitative agreement with MR imaging results using a gadolinium bolus. This result is promising with respect to the potential of bedside monitoring of mean transit time (MTT) changes in patients with stroke or related vascular disease.

We present experimental results on the in vitro and in vivo study of dye diffusion into human skin and hair follicles. We have studied some commercially available dyes for potential using in the laser selective thermolysis. The degree and the depth of hair follicle dyeing inside the skin were determined. For hairs in different stages the sebaceous gland was stated as a reservoir for a dye administration. It was found that the penetration depth of dyes is about 1.2 mm from the skin surface. We have developed the biocompatible Indocyanine Green lotions and the method for in vivo dyeing and dye in depth monitoring. Shift on 16–21 nm of absorption peak of Indocyanine Green to the longer wavelengths due to Indocyanine Green binding with cell proteins in the human skin was found.

A method for the prediction of the average photon pathlength in turbid media has been developed. The method is based on spatially resolved diffuse reflectance with discrete source detector distances up to 2 mm. Light reflectance was simulated using a Monte Carlo technique with a one-layer model utilizing a wide range of optical properties, relevant to human skin. At a source detector separation of 2 mm, the pathlength can vary sixfold due to differences in optical properties. By applying various preprocessing and prediction techniques, the pathlength can be predicted with a root-mean-square error of approximately 5%. Estimation of the photon pathlength can be used, e.g., to remove the influence of optical properties on laser Doppler flowmetry perfusion readings, which are almost linearly related to the average photon pathlength.

The mean time spent by photons inside a nonlocalized optically abnormal embedded inclusion has been derived analytically. The accuracy of the results has been tested against Monte Carlo and experimental data. We show that for quantification of the absorption coefficient of absorptive inclusions, a corrective factor that takes into account the size of the inclusion is needed. This finding suggests that perturbation methods derived for very small inclusions which are used in inverse algorithms require a corrective factor to adequately quantify the differential absorption coefficient of nonlocalized targets embedded in optically turbid media.

The basic mechanisms describing radiation scattered by large optically soft spheres are presented in this paper. The contributions of different components of geometrical optics to formation of the integral indicatrix have been estimated. The asymptotic equations for the asymmetry coefficient have been obtained.

Polymeric microspheres containing a magnetic core have been used in cancer therapy for biophysical targeting of antitumor agents and in magnetic resonance imaging as contrasting agents. For the Human Genome Project, deoxyribose nucleic acid (DNA) capillary electrophoresis has become the most widely used analytical technique where a key component is the design of an effective separation medium. The synthesis and optical characterization of polymeric coated superparamagnetic nanoparticles and of (self-assembled) polymer networks by means of a range of physical techniques, including laser light scattering and laser-induced fluorescence detection, are presented. (1) Polymeric microspheres with a superparamagnetic core. A water-in-oil microemulsion approach has been used successfully to synthesize the superparamagnetic core and the polymeric microsphere in one continuous step. The synthesis permits us to control the magnetic nanoparticle size and the thickness of the hydrogel, ranging from 80 to 320 nm. Magnetite concentration in the microspheres, calculated by vibrating-sample magnetometry, was found to be up to 3.3 wt %. The internal structure of the microspheres, as observed by atomic force microscopy, confirmed a core-shell model. (2) Development of new separation media for DNA capillary electrophoresis. Block copolymers in selective solvents can self-assemble to form supramolecular structures in solution. The nanostructures can be characterized in the dilute concentration regime by means of laser light scattering. At semidilute concentrations, the mesh size, the supramolecular structure, and the surface morphology can be investigated by means of small angle x-ray scattering and atomic force microscopy. The structural knowledge and the information on chain dynamics can then be correlated with electrophoresis using laserinduced fluorescence detection to provide a deeper understanding for the development of new separation media.

cDNA microarrays provide simultaneous expression measurements for thousands of genes that are the result of processing images to recover the average signal intensity from a spot composed of pixels covering the area upon which the cDNA detector has been put down. The accuracy of the signal measurement depends on using an appropriate algorithm to process the images. This includes determining spot locations and processing the data in such a way as to take into account spot geometry, background noise, and various kinds of noise that degrade the signal. This paper presents a stochastic model for microarray images. There are over 20 model parameters, each governed by a probability distribution, that control the signal intensity, spot geometry, spot drift, background effects, and the many kinds of noise that affect microarray images owing to the manner in which they are formed. The model can be used to analyze the performance of image algorithms designed to measure the true signal intensity because the ground truth (signal intensity) for each spot is known. The levels of foreground noise, background noise, and spot distortion can be set, and algorithms can be evaluated under varying conditions.