Morbidity and complexity involved in lymph node staging via surgical resection and biopsy could ideally be overcome using node assay techniques that are non-invasive. Visible blue dyes, fluorophores and radio-tracers are often used to locate the sentinel lymph nodes from draining lymphatic vessels near a tumor, but they only rarely provide an in situ metric to evaluate the presence of cancer. As such, imaging systems that are quantitative and sensitive to surface- and subsurface-fluorescence could provide a radiation-free, less invasive alternative to existing approaches, and have the potential to perform both node mapping and metastasis sensing..;Issues of non-specific uptake, and high delivery variability complicate imaging of single tracers in a lymph node. To overcome these problems, ratiometric schemes using multiple fluorescent tracers along with modeling techniques to estimate cancer biomarkers have recently been demonstrated. In this approach, quantitative estimates of the cancer burden are attainable using micro-doses of fluorescence-labeled tracers targeted to cancer-specific receptors, thus providing high specificity in studying cancer progression and metastasis. In this work, an extensive review of commercial fluorescence imagers and their capabilities led to some unique directions in sensing of lymph nodes with alternative hardware and image processing methods. A previously designed high-frequency ultrasound-guided fluorescence tomography system was upgraded to enable multi-spectral capabilities with large source-detector spacing, to allow subsurface measurements, and the ability to spectrally decouple autofluorescence and signals from multiple fluorophores. Along with tomography, we used planar fluorescence imaging methods to image lymph nodes and lymphatic vessels to quantitatively study lymphatic uptake, flow variation, and lymphatic system changes with tumor progression. In particular, imaging the lymphatic flow in the lymph vessels relative to the uptake in the nodes provided a unique methodology to quantify tumor burden. On the other hand, the value of targeted agents in lymphatic imaging appears to be sensitive to the location and method of delivery, and as such it was found that internal tissue normalization methods were more robust with non-specific fluorophores. Murine models of nodal involvement of metastases, and design of real-time fluorescence imaging tools were combined to propose the most constructive approach to non-invasive quantification of tumor burden in lymph nodes for future clinical implementation.
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