Abstract. Fluorescence spectroscopy has been widely investigated as a technique for identifying pathological tissue; however, unrelated subject-to-subject variations in spectra complicate data analysis and interpretation. We describe and evaluate a new biosensing technique, differential laser-induced perturbation spectroscopy (DLIPS), based on deep ultraviolet (UV) photochemical perturbation in combination with difference spectroscopy. This technique combines sequential fluorescence probing (pre-and post-perturbation) with sub-ablative UV perturbation and difference spectroscopy to provide a new spectral dimension, facilitating two improvements over fluorescence spectroscopy. First, the differential technique eliminates significant variations in absolute fluorescence response within subject populations. Second, UV perturbations alter the extracellular matrix (ECM), directly coupling the DLIPS response to the biological structure. Improved biosensing with DLIPS is demonstrated in vivo in a murine model of chemically induced skin lesion development. Component loading analysis of the data indicates that the DLIPS technique couples to structural proteins in the ECM. Analysis of variance shows that DLIPS has a significant response to emerging pathology as opposed to other population differences. An optimal likelihood ratio classifier for the DLIPS dataset shows that this technique holds promise for improved diagnosis of epithelial pathology.Results further indicate that DLIPS may improve diagnosis of tissue by augmenting fluorescence spectra (i.e. orthogonal sensing).

Vascular targeting agents on their own have been shown to be insufficient for complete treatment of solid tumors, emphasizing the importance of studying the vascular effects of these drugs for their use with conventional therapies in the clinic. First-pass fluorescence imaging combined with hyperspectral imaging of hemoglobin saturation of microvessels in the murine dorsal window chamber model provides an easily implementable, low cost method to analyze tumor vascular response to these agents in real-time. In this study, the authors utilized these methods to spectroscopically demonstrate distinct vessel structure, blood flow and oxygenation changes in human Caki-2 renal cell carcinoma following treatment with OXi4503 alone, Sunitinib alone and both drugs together. We showed that treatment with OXi4503 plus Sunitinib destroyed existing tumor microvessels, inhibited blood vessel recovery and impaired Caki-2 tumor growth significantly more than either treatment alone.

Hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence imaging of blood transit time were combined to analyze the oxygenation of and blood flow through microvessel networks. The combination imaging technique was demonstrated in a mouse dorsal window chamber model of a growing Caki-2 human renal cell carcinoma over time. Data from Hb saturation and blood supply time maps show the formation of arteriovenous malformations and shunting of blood directly from arteries to the tumor core and into veins in the periphery of the tumor. Images and data analysis show these malformations result in an oxygenated environment ideal for a tumor to proliferate.

We have set the stage for a feasibility demonstration using a novel form of testing. With sufficient subjects and proper statistical evaluation, a robust test method for assessing the performance of pulse oximeters in the presence of motion may be at hand.

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