Spectral imaging techniques, using visible and near-infrared radiation, are being investigated for their potential to detect abnormal regions deeply embedded within normal tissue. If successful, these techniques offer a variety of functional imaging modalities in addition to density imaging, while avoiding ionizing radiation hazards. One of the most challenging areas to apply diffuse optical imaging of deep tissues is the human breast. We have devised a new approach that uses time-dependent contrast functions, based on a random walk theory on a lattice, to derive the optical properties and the size of an abnormal target from TOF data measured in time-resolved transillumination experiments. We validated our methodology in phantom experiments. Recently, we have applied our method to quantify optical properties of breast tumors for several patients presenting with invasive ductal carcinoma. The tumors showed increased absorption and scattering. From the absorption coefficients at both wavelengths, blood oxygen saturation was estimated for the tumors and the surrounding tissue. We found that the tumors are hypoxic and their blood volume is increased by about a factor of two in comparison with surrounding tissue, indicating increased vascularization. We are continuing to analyze additional in-vivo breast measurements. We are planning to use this technique in clinical protocols at NCI designed to test neo-adjuvent drugs. The development of fluorescently labeled cell surface markers has opened the possibility of specific and quantitative noninvasive diagnosis of tissue changes. We are developing a fluorescence scanning imaging system that can perform a ?noninvasive optical biopsy?. We have derived an exact mathematical expression for fluorescent signals emitted from an optically turbid medium containing fluorescent masses, showing how the signal depends on the many parameters which are involved (absorption and scattering coefficients at the excitation and emission wavelengths, depth, quantum yield, etc?). We envision an instrument in which a laser scans the tissue surface, yielding a sequence of images corresponding to each surface point irradiated, from which we would compute the 3-D concentration distribution of the fluorophore. We have successfully tested the inverse algorithm that underlies our analysis by simulating data from one and two fluorophores embedded at different depths and different distances relative to each other. Even when the distance between the two fluorophores is such to provide a single peak, we were able to localize and determine the relative strength of the fluorophore masses with good accuracy. We plan to test this combined image acquisition and analysis system in vitro with tissue phantoms containing known embedded fluorescent objects. The results of the analysis of the image acquisition by our system will be evaluated for its clinical utility by direct comparison with subsequent pathology of the same tissue. We are heavily involved in the use of optical molecular contrast agents. As an example, we have started a series of animal experiments. In these experiments, the tongues of Balb-C mice are injected with Squamous Carcinoma Cells that are CD3 and CD19 positive. FITCI conjugated antibodies to CD3 or CD19 are then injected into the tongue. Our goal is to study whether our model of diffuse fluorescent photon migration is able to separate the effects of light diffusion at a given depth from the actual distributions of the fluorescent antibodies. The development of semiconductor quantum dots for biological applications has provided a new class of fluorophores that are extremely resistant to photobleaching, have readily tunable narrow-band fluorescence, and are of a size (5-10 nm diameter) and stability to be especially useful in pharmacokinetic studies. Using BSA-coated CdMnTe/Hg quantum dots, we have demonstrated that the nanocrystals are a useful angiogrraphic contrast agent in mice. The nanocrystals were administered via jugular injection; the excitation source was a 633 nm He-Ne laser, and the fluorescence, peaked around 775 nm, was captured using a sensitive CCD camera with a bandpass filter. Preliminary assessment of the depth of penetration for excitation and emission was done by imaging a beating mouse heart, both through an intact thorax and after a thoracotomy. The temporal resolution associated with imaging the nanocrystals in circulation has been addressed, and the blood clearance for this contrast agent has also been measured. In addition, we directly imaged the vessels surrounding and penetrating a murine squamous cell carcinoma in a C3H mouse. These preliminary results suggest that the NIR-fluorescent quantum dots may be ideally suited for pharmacokinetic studies. Development of new drugs targeting angiogenetic activity or blood flow have brought the need to non-invasively monitor blood circulation. We are investigating the use of three imaging modalities to quantify different parameters associated with blood circulation. These are: 1) Thermography, which provides a two-dimensional image of superficial skin temperatures (the concept is that higher temperatures occur in the skin superficial to veins that are involved in active transport of blood) 2) Laser Doppler Imaging (LDI) which produces two dimensional images of blood flow over a defined area at 690nm and 780nm; and 3) Multi-spectral imaging, which produces two dimensional reflectance images at six wavelengths (700-1000nm, increment of 50nm). For the latter method we have developed an algorithm which separate the effects of melanin absorption, and light scattering in order to provide a map of oxy- deoxy-hemoglobin ratio (i.e., oxygenation) and total blood content under the skin. Although these three modalities are semi-quantitative, each gives different parameters related to blood flow. Our goal is to cross-correlate results from these modalities and assess blood circulation. We are participating in an NCI sponsored clinical trial evaluating the effectiveness of anti-angiogenetic drug treatment for Kaposi?s Sarcoma (KS). (KS) is a highly vascular tumor. Angiogenesis and capillary permeability can play an important role in the development and progression of KS. Yet no non-invasive standard technique currently is available to assess the effect of anti-angiogenetic therapy on blood flow to the tumors. The purpose of the clinical trial is to investigate the applicability of the three non-invasive methods for the assessment of vascularity and vascular changes associated with KS and ultimately predict the outcome of the treatment. Before applying an anti-angiogenesis drug, a comparative image analysis of thermography, LDI, and multi-spectral images are taken from the lesion and the contralateral, lesion free sites. Six patients have already finished the first phase of the treatment. Preliminary results obtained from these three modalities show that lesions with higher temperature and blood flow compared to contralateral side are improved after treatment, whereas, based on conventional biopsy, cooler lesions are not responding to the treatment. No definitive assessments were possible based on blood volume and oxygenation. Preliminary results indicate that the techniques may be useful to assess vascularity and vascular changes associated with KS, and monitor the outcome of the treatment. Completion of the ongoing study should provide additional data to support these initial results.