The Optical Imaging Laboratory has developed a hand-held optical imaging system that is capable of 3D tomographic imaging. However, the imaging system is limited by longer imaging times, and inaccuracy in the positional tracking of the hand-held probe. Hence, the objective is to improve the performance of the imaging system by improving imaging time and positional accuracy. This involves: (i) development of automated single Labview-based software towards near real-time imaging; and (ii) implementation of an alternative positional tracking device (optical) towards improved positional accuracy during imaging. Experimental studies were performed using cubical tissue phantoms (1% Liposyn solution) and 0.45-cc fluorescence target(s) placed under various conditions. The studies demonstrated a 90% reduction in the imaging time (now ~27 sec/image) and also an increase from 94% to 97% in the positional accuracy of the hand-held probe. Performance enhancements in the hand-held optical imaging system have improved its potential towards clinical breast imaging.

No / Lymphocyte responses from 208 individuals: 20 with melanoma, 34 with colon cancer, and 4 with lung cancer (58), 18 with suspected melanoma, 28 with polyposis, and 10 with COPD (56), and 94 healthy volunteers were examined. The natural logarithm of the Olive tail moment (OTM) was plotted for exposure to UVA through 5 different agar depths (100 cell measurements/depth) and analyzed using a repeated measures regression model. Responses of patients with cancer plateaued after treatment with different UVA intensities, but returned toward control values for healthy volunteers. For precancerous conditions and suspected cancers, intermediate responses occurred. ROC analysis of mean log OTMs, for cancers plus precancerous/suspect conditions vs. controls, cancer vs. precancerous/suspect conditions plus controls, and cancer vs. controls, gave areas under the curve of 0.87, 0.89, and 0.93, respectively (P<0.001). Optimization allowed test sensitivity or specificity to approach 100% with acceptable complementary measures. This modified comet assay could represent a stand-alone test or an adjunct to other investigative procedures for detecting cancer.

To confirm the presence of prostate cancer which is the most incident visceral cancer in men, prostate biopsies are acquired using the magnetic resonance imaging fusion-guided prostate biopsy protocol. For this approach annotated magnetic resonance imaging is overlaid onto real-time ultrasound imaging to guide sampling of suspicious regions marked by uroradiologists. Additional biopsy samples are acquired via the previous clinical gold standard, i.e. the templated 12-core transrectal ultrasound-guided prostate biopsy protocol. While this approach improves the sensitivity of the prostate biopsy, a real-time, multiparametric imaging method of identifying biopsy targets could help overcome some of the inherent pitfalls of the magnetic resonance imaging fusion-guided prostate biopsy. Since ultrasound is used during the prostate biopsy, photoacoustic tomography, e.g. a hybrid imaging modality in which clinical ultrasound probes can be used to detect centimeters deep chemical alterations, has the potential to provide real-time targeting during biopsy. The translation of photoacoustic tomography to the clinic for prostate biopsy has been prevented by engineering challenges, which include identification of a biomarker for detecting suspicious regions of tissue and light delivery to the prostate for photoacoustic signal generation. Here, we present a vascular texture analysis method that identified 100% of primary and 67% of secondary tumors in the testing data set of ex vivo human prostate specimens. This method can be applied to future in vivo photoacoustic and ultrasound tomography of human prostates after further optimization of light delivery for photoacoustic tomography. To progress towards achieving this aim, we developed a transurethral light delivery device with angular light coupling method. By controlling the launch angle of the light into the fiber, the conversion of forward to side propagating energy can be improved from 27% to 98%, and the longitudinal emission profile can be controlled in order to illuminate the whole prostate simultaneously.<br>

Breast cancer is the most common cancer in women worldwide. In 2009, a novel imaging modality called Shear Wave Elastography (SWE), an ultrasound technique visualising the elasticity of tissue, was introduced to the field of clinical breast imaging. Because malignant tissues are generally stiffer than benign tissues, SWE supports the differentiation of benign / malignant solid breast lesions. However, no standard has yet been defined for the application and the evaluation of results. Furthermore, image evaluation has to be carried out directly from the ultrasound system, complicating long-term and multi-centre studies. This PhD thesis investigated the influences from the imaging process and image evaluation on SWE measurements. Various parameters were appraised with regard to their diagnostic performance, in order to define the best clinical standard. To define more complex image analysis, taking the parameters investigated into account, algorithms were devised to enable automatic assessment of B-mode and SWE images. In this work, influences from the imaging process and image evaluation on the SWE measurements were demonstrated. The influences investigated included: the impact from the region of interest and the imaging plane used; the individual variation in breast composition; the number of images considered and the pressure applied during imaging. The algorithms described within this work achieved a diagnostic accuracy similar to that of manual assessment by a radiology expert. This thesis demonstrated influences from the imaging process and image evaluation on the SWE measurements obtained. Taking these influences into consideration would complicate the clinical application of SWE imaging. However, automatic image evaluation as presented here would overcome this issue. Using the guidelines defined in this PhD thesis also allows for comparison of results taken from different imaging sites.

The clinical management of glioma remains a challenge. The prognosis is poor—for glioblastoma multiforme, the most virulent of these brain cancers, survival is only ~1 year. Surgical resection of the tumor is the first line of defense. Several studies demonstrate a survival advantage in patients who undergo near-complete tumor resection; however, achieving complete resection is limited by the difficulty of visualizing residual tumor after de-bulking. Intraoperative fluorescence guidance is a promising candidate to better visualize residual tumor. The most clinically developed form uses protoporphyrin IX fluorescence, the precursor to heme in its biosynthesis which preferentially accumulates in tumor cells after the administration of 5-aminolevulinic acid. Challenges remain in quantitatively assessing the fluorescence to reduce variability of outcome and improve tumor detection specificity, and in observing sub-surface tumor fluorescence. To these ends, this work outlines the development of intraoperative techniques to 1) quantify tissue fluorescence using a handheld fiberoptic probe and 2) improve detection by reconstructing the depth-resolved fluorescence topography of sub-surface tumor. As a critical component to achieve these objectives, a technique to measure the tissue optical properties was developed. This technique used diffuse reflectance measurements mediated by a handheld fiberoptic probe to derive the tissue optical properties. The handheld fiberoptic probe was further developed to include fluorescence spectroscopy. A novel algorithm to combine the fluorescence measurement and the tissue optical properties was derived in order to extract the quantitative fluorescence spectrum, i.e. fluorescence without confounding effects of tissue optical properties. The concentration of fluorescent tumor biomarker can then be extracted. The quantitative fluorescence work culminated in deployment of the fiberoptic probe in clinical trials for the resection of intracranial tumors. The quantitative fluorescence probe out-performed a state-of-the-art fluorescence surgical microscope for a broad range of brain tumor pathologies. A novel technique for depth-resolved fluorescence detection was developed utilizing multi-excitation fluorescence imaging. An algorithm to extract depth information from the multi-excitation images was derived, with validation in phantoms and a rat brain tumor model. This demonstrates the potential for depth-resolved fluorescence imaging, which there is a clear need for in tumor resection guidance.

The clinical management of glioma remains a challenge. The prognosis is poor—for glioblastoma multiforme, the most virulent of these brain cancers, survival is only ~1 year. Surgical resection of the tumor is the first line of defense. Several studies demonstrate a survival advantage in patients who undergo near-complete tumor resection; however, achieving complete resection is limited by the difficulty of visualizing residual tumor after de-bulking. Intraoperative fluorescence guidance is a promising candidate to better visualize residual tumor. The most clinically developed form uses protoporphyrin IX fluorescence, the precursor to heme in its biosynthesis which preferentially accumulates in tumor cells after the administration of 5-aminolevulinic acid. Challenges remain in quantitatively assessing the fluorescence to reduce variability of outcome and improve tumor detection specificity, and in observing sub-surface tumor fluorescence. To these ends, this work outlines the development of intraoperative techniques to 1) quantify tissue fluorescence using a handheld fiberoptic probe and 2) improve detection by reconstructing the depth-resolved fluorescence topography of sub-surface tumor. As a critical component to achieve these objectives, a technique to measure the tissue optical properties was developed. This technique used diffuse reflectance measurements mediated by a handheld fiberoptic probe to derive the tissue optical properties. The handheld fiberoptic probe was further developed to include fluorescence spectroscopy. A novel algorithm to combine the fluorescence measurement and the tissue optical properties was derived in order to extract the quantitative fluorescence spectrum, i.e. fluorescence without confounding effects of tissue optical properties. The concentration of fluorescent tumor biomarker can then be extracted. The quantitative fluorescence work culminated in deployment of the fiberoptic probe in clinical trials for the resection of intracranial tumors. The quantitative fluorescence probe out-performed a state-of-the-art fluorescence surgical microscope for a broad range of brain tumor pathologies. A novel technique for depth-resolved fluorescence detection was developed utilizing multi-excitation fluorescence imaging. An algorithm to extract depth information from the multi-excitation images was derived, with validation in phantoms and a rat brain tumor model. This demonstrates the potential for depth-resolved fluorescence imaging, which there is a clear need for in tumor resection guidance.

Molecular analysis of free circulating (fc)DNA has the potential to change the face of medicine, specifically in cancer diagnostics and in monitoring the efficacy of cancer treatments. In this study, a microfluidic device using AC electrokinetics is developed for rapid concentration and detection of fcDNA from blood. The device concentrates fcDNA using a combination of AC electrothermal flow and dielectrophoresis. The electrothermal fluid motion drives fcDNA towards the center of the electrode where dielectrophoretic trapping occurs. Once fcDNA is collected at the center, the concentration in the sample can be determined by fluorescent analysis using an intercalating dye binding to the double-stranded DNA. Effects of operating parameters are investigated to optimize the device's design. The electrokinetic device isolates high molecular weight DNA and can distinguish from low molecular weight DNA. Quantitative detection of fcDNA in physiologically relevant concentrations is demonstrated toward rapid diagnostics of cancer and monitoring of treatment efficacy.

Doctor of Philosophy / Department of Chemistry / Stefan Bossmann / Numerous Proteases are implicated in cancer initiation, survival, and progression. Therefore, it is important to diagnose the levels of protease expression by tumors and surrounding tissues, which are reflected in blood and tissue samples. Nanoplatforms for Cathepsin(CTS) B and L, matrix metalloproteinases(MMP) 1, 2, 3, 7, 9, 13 and urokinase plasminogen activator(uPA) detection have been synthesized. Nanoplatforms feature a central dopamine-coated core/shell Fe/Fe₃O₄ nanoparticle. Cyanine 5.5 is permanently tethered to the dopamine ligands via amide bonds. Tetrakis(4-carboxy-phenyl)porphyrin (TCPP) is co-tethered to Fe/Fe₃O₄/dopamine by means of protease consensus sequences. In the presence of a relevant protease sequence, it is cleaved, releasing TCPP from the nanoplatform. In contrast, Cy 5.5 will remain permanently tethered to the nanoparticle. Therefore, an extensive increase of emission intensity of the fluorescence signal from TCPP is observed. This permits the detection of the activity of proteases at femtomolar levels in biospecimens by fluorescence spectroscopy. 46 breast cancer and 20 healthy human blood serum samples were analyzed. Based on the expression pattern of analyzed enzymes, human breast cancer can be detected at stage I. By monitoring CTS B and L stage 0 detection may be achieved. This study demonstrates the feasibility of minimally invasive successful early cancer diagnosis. Immunosuppression is one of the hallmarks of aggressive cancers. Arginase is overexpressed in cancer patients, resulting in systemic immunosuppression. Two nanoplatforms for arginase detection have been synthesized. Both feature a central dopamine-coated core/shell Fe/Fe₃O₄ nanoparticle to which cyanine 7.0 or cyanine 7.5 is tethered via amide bonds. In both nanoplatforms, cyanine 5.5 is linked to the N-terminal of the peptide sequence GRRRRRRRG. Arginine (R) reacts to ornithine (O) in the presence of arginase. According to our results obtained from fluorescence spectroscopy, the oligopeptides GRRRRRRRG and GOOOOOOOG differ in their chain dynamics. In the presence of arginase, and dependent on arginase activity, fluorescence increase of both nanoplatforms is observed, which is an indication that proton-transfer quenching decreases when arginine gets converted to ornithine. The novel assays permit the detection of active arginase within an hour. Additionally, Förster Resonance Energy Transfer (FRET) is observed in nanoplatforms featuring cy 5.5/7.0 pairs, resulting in picomolar detection limits. This is the first example of a “post-translational” enzyme sensor, in which the tether is subjected to chemical transformations of the aminoacid side chains and not cleaved by an enzyme, resulting in the modified mobility of the tether. The nanoplatforms do not show a fluorescence increase when incubated with NO-reductase, an enzyme indicative of immunoactivation, which also uses arginase as substrate. Copper dependent inhibitory activity of 10000 compound library has been studied against of Staphylococcus aureus. 53 copper- dependent hit molecules were recognized featuring extended thiourea core structure with NNSN motif. NMR titrations, UV/Vis studies have been performed for characterization of metal complexation and structure modeling. Chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, in spite of other compound groups in chemical screening libraries. This will lead to the development of novel class of antibacterial agents against Staphylococcus aureus.

Cancer is one of the leading cause of death in modern world, so there is an emerging demand for better diagnostic tools and more specific less toxique therapeutics. Nanoparticles offers characteristics that could fullfill such perspectives. They can easily target tumor by ehanced permeation and retention effect (EPR). Nanoparticles can combine more than one imaging properties, so we can say that they are multimodal, some of them could combine diagnostic and therapeutic molecules in one nanoparticle, which is now highly popular topic of nanoparticles for theranostics . The aim of this thesis was to characterize new multimodal glycogen-based nanoparticle. Glycogen is an ideal structure for nanoparticle design. Glycogen is part of natural dendrimers group which are easily to modify. Glycogen's size is suitable for EPR effect. We have evaluated biological characteristics of five different types of modified glycogen. The in vitro experiments were carried on HepG2 cells. We have set time curve of cellular uptake of this glycogen probes, evaluated cytoplasmatic localization and for the first time we have carried MTT assay. Biodistribution studies on CD1-Nude mice were performed by using non-invasive method for measuring in vivo fluorescence. In conlusion we've provided some of the biological characteristics of new...

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