Optical imaging through biological tissues and other scattering media is challenging, as the scattered light creates an extremely high background noise level that makes it difficult to detect objects that are embedded within the media. This thesis examines a relatively unexplored method of separating scattered light from unscattered light that has application to optical imaging through turbid media. The method creates an optical filter that blocks photons based upon their exit trajectory direction. Such a trajectory filter can be used with a collimated beam that transmissively illuminates a scattering medium to create an imaging system in which a shadowgram is formed from those photons that pass through the filter and have a trajectory close to that of the collimated beam. Experiments have shown that such a system is effective up to measured optical depths of 18 to 21 and scattering ratios of 108 to 109 using both coherent and incoherent sources. A micromachined linear array of 50 m x 10 mm collimating holes was developed earlier as a photon trajectory filter and was used to successfully image through media in which the ratio of scattered to unscattered light is extremely high (>107). These results are much better than simple theory would predict. This thesis provides a theoretical basis for the trajectory filter system to allow its performance to be characterized and compared against other optical imaging methods, such as time-domain imaging. Using Monte Carlo simulations, it is found that the trajectory filter method is more effective than pathlength-based methods for imaging through turbid media with moderate levels of scattering, up to ̃optical depths, and that it can be combined with other imaging methods to further improve contrast. Advantages of the trajectory filter method include coherence and wavelength invariance and the ability to perform either wide beam, full-field or narrow beam, scanned imaging. Experimental results are presented for laser and incoherent beams using two types of trajectory filters: spatiofrequency and linear collimating hole array. It is found that the trajectory filter method offers a viable means of transmissively imaging through moderately scattering media at optical and near infrared wavelengths.

This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The authors have demonstrated the use of a degenerate-four-wave-mixing time gate to allow imaging through turbid media, with potential application to tissue imaging. A near infrared (NIR), long-pulse Cr{sup +3}:Li2SrAlF6 laser was used as the light source (during most the project) for imaging through clear and turbid media. Preliminary experiments were also carried out with a continuous diode laser.

Experiments directed towards a clinically useful optical imaging system use long-pulse near-infrared lasers and a correlation time gate based on degenerate four-wave mixing in a nonlinear medium.

Assessment of anisotropy has many applications in tissue engineering and early detection of disease such as cancer or stroke. One of the emerging optical technologies for quantifying anisotropy in biological tissues is polarized light imaging. In this technique, the full Mueller matrix of the tissue is measured and then decomposed to yield optical retardance, which is proportional to the tissue anisotropy. The theory of polarized light imaging, its biomedical application and instrumentation are the subject of this thesis. To retrieve more information from the Mueller matrix, a quantitative analysis of generic turbid media's Mueller matrices beyond their decompositions was proposed. Two new metrics based on Mueller matrix were established in this thesis: 1) Tissue depolarization, calculated from the Mueller matrix, can be used to estimate its optical properties, and 2) Symmetry of the off-diagonal elements of a turbid medium's Mueller matrix can be used to detect the axial heterogeneity of anisotropy. Polarized light imaging, followed by Mueller matrix decomposition, was then successfully applied to locate the structural disorders induced by bladder outlet obstruction disease, in ex vivo functioning rat bladders. Motivated by the result of this study and to enable in vivo polarized light measurements of the bladder, a novel thin fiber based polarimetric probe which can measure the full Mueller matrix of the turbid media was suggested. Finally, to enhance the speed of the ex vivo examinations of biological tissues and avoid artifacts, a new rapid benchtop polarized light imaging system based on photoelastic modulators (PEM) and a charge-coupled device (CCD) was proposed and implemented. The demonstrated scheme does not involve using mechanically moving components and thus reduces systematic errors. This imaging system proved to be the fastest high resolution polarimetric characterization tool for imaging turbid media to date.

Modulated Imaging (MI) is a fast, scan-free method that enables one to image and quantify the optical properties of turbid media. The technology can simultaneously map surface and sub-surface tissue structure, function and composition. Based on frequency-domain measurement principles, MI uses spatially-periodic or "structured" illumination and camera-based detection to separate and quantify the absorption, scattering, and fluorescence optical properties over a wide field-of-view (many cm) without the need for sample contact. Resolution is depth-dependent and thus scalable (sub-millimeter to millimeter), with depth sensitivity up to a few cm. This method has particularly strong potential for in-vivo clinical and pre-clinical imaging, where optical properties at several wavelengths provide quantitative information on endogeneous chromophore concentrations (i.e. oxy- and deoxy-hemoglobin, fat, and water). These parameters reflect quantitative, localized tissue status such as blood volume, tissue oxygenation, and edema. Using multispectral MI instrumentation, demonstrations of two in-vivo applications are investigated: (1) pre-clinical functional imaging of brain injury in a rodent model and (2) clinical imaging spectroscopy of human skin. Also, preliminary 3D fluorescence tomography data suggest that MI may provide a convenient, low-cost platform for localizing and quantifying exogenous molecular probes in-vivo.

"Nonlinear optical biomedical imaging techniques have attracted a great amount of interest starting with the first demonstration of two-photon fluorescence (TPF) imaging by Webb's group in the 1990s. The various imaging formats and applications reported have revolutionized the field of biomedical imaging, offering great advantages including high resolution, internal three-dimensional sectioning ability, and functional imaging capability. This thesis focuses on exploring the development and novel application of nonlinear optical techniques and extending previous work in transmission mode nonlinear absorption imaging. The topical pharmacological introduction of molecules and drugs to the ocular surface is a common means of assessing and treating a variety of pathological ocular conditions. However, no reliable method exists to date for the quantification of actual delivered dosage with high enough resolution. We developed a two-photon fluorescence (TPF) system capable of quantifying, with micron-level axial resolution, the distribution and concentration of fluorescent or fluorescently-tagged chemicals and drugs in live feline corneas. With this high-resolution method, we were able to measure, for the first time, both the penetration depth and concentrations of molecules applied topically to the ocular surface, either with an intact or removed epithelial layer. As a proof of concept, we tested two classes of fluorescent molecules- Fluorescein and Riboflavin- which are commonly used in ophthalmologic practice. Finally, we used our TPF instrument to test the barrier function of the corneal epithelium and to measure the concentration of non-fluorescent molecules (in this case, dextrans) conjugated to fluorescent dyes as they diffused across the cornea. A pump-probe based technique has been applied in biomedical imaging by Prof. Warren Warren's group recently. They reported images with endogenous contrast agents (hemoglobin and melanin) in biological tissue in a transmission mode with two pulsed laser systems to generate two wavelengths for the pump and probe beam. We built two simplified systems with only one Ti:Sapphire laser. In both systems, the pump and probe beam were selected from a broadband source, which was generated by either broadening the spectrum with a holey fiber or a 27 fs KM laser, which has a broad spectrum itself. We explored the capability of imaging in tissue-like turbid media in the backscattering mode, and studied the achievable imaging depth for the first time. By simulating using Monte Carlo based methods, we further optimized the detection geometry and improved the photon collection efficiency. Also, we compared this nonlinear absorption technique with the more commonly used TPF method. We finally obtained pump probe signals and images using quantum dots as a nonlinear medium. This could be important in future studies of toxicity in skin-care products"--Leaves vi-vii.