Jennifer M. Watson

In Vivo, Dual-Modality OCT/LIF Imaging Using a Novel VEGF Receptor-Targeted NIR Fluorescent Probe in the AOM-Treated Mouse Model

PurposeIncreased vascular endothelial growth factor (VEGF) receptor expression has been found at the sites of angiogenesis, particularly in tumor growth areas, as compared with quiescent vasculature. An increase in VEGF receptor-2 is associated with colon cancer progression. The in vivo detection of VEGF receptor is of interest for the purposes of studying basic mechanisms of carcinogenesis, making clinical diagnoses, and monitoring the efficacy of chemopreventive and therapeutic agents. In this study, a novel single chain (sc)VEGF-based molecular probe is utilized in the azoxymethane (AOM)-treated mouse model of colorectal cancer to study delivery route and specificity for disease.ProceduresThe probe was constructed by site-specific conjugation of a near-infrared fluorescent dye, Cy5.5, to scVEGF and detected in vivo with a dual-modality optical coherence tomography/laser-induced fluorescence (OCT/LIF) endoscopic system. A probe inactivated via excessive biotinylation was utilized as a control for nonreceptor-mediated binding. The LIF excitation source was a 633-nm He:Ne laser, and red/near-infrared fluorescence was detected with a spectrometer. OCT was used to obtain two-dimensional longitudinal tomograms at eight rotations in the distal colon. Fluorescence emission levels were correlated with OCT-detected disease in vivo. OCT-detected disease was verified with hematoxylin and eosin stained histology slides ex vivo.ResultsHigh fluorescence emission intensity from the targeted probe was correlated with tumor presence as detected using OCT in vivo and VEGFR-2 immunostaining on histological sections ex vivo. The inactivated probe accumulated preferentially on the surface of tumor lesions and in lymphoid aggregate tissue and was less selective for VEGFR-2.ConclusionThe scVEGF/Cy probe delivered via colonic lavage reaches tumor vasculature and selectively accumulates in VEGFR-2-positive areas, resulting in high sensitivity and specificity for tumor detection. The combination of OCT and LIF imaging modalities may allow the simultaneous study of tumor morphology and protein expression for the development of diagnostic and therapeutic methods for colorectal cancer.

Analysis of second-harmonic-generation microscopy in a mouse model of ovarian carcinoma

Second-harmonic-generation (SHG) imaging of mouse ovaries ex vivo was used to detect collagen structure changes accompanying ovarian cancer development. Dosing with 4-vinylcyclohexene diepoxide and 7,12-dimethylbenz[a]anthracene resulted in histologically confirmed cases of normal, benign abnormality, dysplasia, and carcinoma. Parameters for each SHG image were calculated using the Fourier transform matrix and gray-level co-occurrence matrix (GLCM). Cancer versus normal and cancer versus all other diagnoses showed the greatest separation using the parameters derived from power in the highest-frequency region and GLCM energy. Mixed effects models showed that these parameters were significantly different between cancer and normal (P<0.008). Images were classified with a support vector machine, using 25% of the data for training and 75% for testing. Utilizing all images with signal greater than the noise level, cancer versus not-cancer specimens were classified with 81.2% sensitivity and 80.0% specificity, and cancer versus normal specimens were classified with 77.8% sensitivity and 79.3% specificity. Utilizing only images with greater than of 75% of the field of view containing signal improved sensitivity and specificity for cancer versus normal to 81.5% and 81.1%. These results suggest that using SHG to visualize collagen structure in ovaries could help with early cancer detection.

Two-photon excited fluorescence imaging of endogenous contrast in a mouse model of ovarian cancer†‡

Ovarian cancer has an extremely high mortality rate resulting from poor understanding of the disease. In order to aid understanding of disease etiology and progression, we identify the endogenous fluorophores present in a mouse model of ovarian cancer and describe changes in fluorophore abundance and distribution with age and disease.

In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis

Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SHG]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a long-term survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SHG. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer.

Sequential compression devices in postoperative urologic patients: an observational trial and survey study on the influence of patient and hospital factors on compliance

BackgroundSequential compression devices (SCDs) are commonly used for thromboprophylaxis in postoperative patients but compliance is often poor. We investigated causes for noncompliance, examining both hospital and patient related factors.Methods100 patients undergoing inpatient urologic surgery were enrolled. All patient had SCD sleeves placed preoperatively. Postoperative observations determined SCD compliance and reasons for non-compliance. Patient demographics, length of stay, inpatient unit type, and surgery type were recorded. At discharge, a patient survey gauged knowledge and attitudes regarding SCDs and bother with SCDs. Statistical analysis was performed to correlate SCD compliance with patient demographics; patient knowledge and attitudes regarding SCDs; and patient self-reported bother with SCDs.ResultsObserved overall compliance was 78.6%. The most commonly observed reasons for non-compliance were SCD machines not being initially available on the ward (71% of non-compliant observations on post-operative day 1) and SCD use not being restarted promptly after return to bed (50% of non-compliant observations for entire hospital stay). Mean self-reported bother scores related to SCDs were low, ranging from 1–3 out of 10 for all 12 categories of bother assessed. Patient demographics, knowledge, attitudes and bother with SCD devices were not significantly associated with non-compliance.ConclusionsPatient self-reported bother with SCD devices was low. Hospital factors, including SCD machine availability and timely restarting of devices by nursing staff when a patient returns to bed, played a greater role in SCD non-compliance than patient factors. Identifying and addressing hospital related causes for poor SCD compliance may improve postoperative urologic patient safety.

Simultaneous multiplane imaging of human ovarian cancer by volume holographic imaging

Abstract. Ovarian cancer is the most deadly gynecologic cancer, a fact which is attributable to poor early detection and survival once the disease has reached advanced stages. Intraoperative laparoscopic volume holographic imaging has the potential to provide simultaneous visualization of surface and subsurface structures in ovarian tissues for improved assessment of developing ovarian cancer. In this ex vivo ovarian tissue study, we assembled a benchtop volume holographic imaging system (VHIS) to characterize the microarchitecture of 78 normal and 40 abnormal tissue specimens derived from ovarian, fallopian tube, uterine, and peritoneal tissues, collected from 26 patients aged 22 to 73 undergoing bilateral salpingo-oophorectomy, hysterectomy with bilateral salpingo-oophorectomy, or abdominal cytoreductive surgery. All tissues were successfully imaged with the VHIS in both reflectance- and fluorescence-modes revealing morphological features which can be used to distinguish between normal, benign abnormalities, and cancerous tissues. We present the development and successful application of VHIS for imaging human ovarian tissue. Comparison of VHIS images with corresponding histopathology allowed for qualitatively distinguishing microstructural features unique to the studied tissue type and disease state. These results motivate the development of a laparoscopic VHIS for evaluating the surface and subsurface morphological alterations in ovarian cancer pathogenesis.

Diagnostic potential of multimodal imaging of ovarian tissue using optical coherence tomography and second-harmonic generation microscopy.

Abstract. Ovarian cancer is particularly deadly because it is usually diagnosed after it has metastasized. We have previously identified features of ovarian cancer using optical coherence tomography (OCT) and second-harmonic generation (SHG) microscopy (targeting collagen). OCT provides an image of the ovarian microstructure, while SHG provides a high-resolution map of collagen fiber bundle arrangement. Here, we investigated the diagnostic potential of dual-modality OCT and SHG imaging. We conducted a fully crossed, multireader, multicase study using seven human observers. Each observer classified 44 ex vivo mouse ovaries (16 normal and 28 abnormal) as normal or abnormal from OCT, SHG, and simultaneously viewed, coregistered OCT and SHG images and provided a confidence rating on a six-point scale. We determined the average receiver operating characteristic (ROC) curves, area under the ROC curves (AUC), and other quantitative figures of merit. The results show that OCT has diagnostic potential with an average AUC of 0.91±0.06. The average AUC for SHG was less promising at 0.71±0.13. The average AUC for simultaneous OCT and SHG was not significantly different from OCT alone, possibly due to the limited SHG field of view. The high performance of OCT and coregistered OCT and SHG warrants further investigation.

Adjustable nanomanufacturing using template-guided self-assembly

Template-guided self-assembly is one of the most promising approaches for creating functional nanosystems and effective methods are needed in order to generate micro and nanoscale templates for a diverse type of materials necessary for different applications. In this paper a plasma lithography technique is demonstrated which can directly pattern a wide variety of substrates including polystyrene, PDMS and glass for self-assembly of nanomaterials. The technique employs a deformable mold made using PDMS replication molding. The mold protects selective areas of the substrate from plasma surface treatment, which will spatially alter the surface properties of the substrate. Nanomaterials will then selectively self-assemble onto the pattern. Three-dimensional control is achieved by means of mold geometry and self-assembly conditions. Nanomaterials including nanoparticles, proteins, and salts have been patterned on configurations from arrays of lines and dots to arbitrary shapes in sizes ranging from millimeters to hundreds of nanometers.

Objective assessment of multimodality optical coherence tomography and second-harmonic generation image quality of ex vivo mouse ovaries using human observers

Ovarian cancer is particularly deadly because it is usually diagnosed after it has begun to spread. Transvaginal sonography (TVS) is the most common imaging screening technique. However, routine use of TVS has not reduced ovarian cancer mortality. The superior resolution of optical imaging techniques may make them attractive alternatives to TVS. We have previously identified features of ovarian cancer using optical coherence tomography (OCT) and secondharmonic generation (SHG) microscopy (with collagen as the targeted fluorophore). OCT provides a gross anatomical image of the ovary while SHG provides a closer look at a particular region. Knowing these anatomical features, we sought to investigate the diagnostic potential of OCT and SHG. We conducted a fully crossed, multi-reader, multi-case study using seven human observers. Each observer classified 44 ex vivo mouse ovaries as normal or abnormal from OCT, SHG, and simultaneous, co-registered OCT and SHG images and provided a confidence rating on a three-point ordinal scale. We determined the average receiver operating characteristic (ROC) curves, area under the ROC curves (AUC), and other quantitative figures of merit. The results show that OCT has diagnostic potential with an average AUC of 0.91 ± 0.03. The average AUC for SHG was less promising at 0.71 ± 0.06. Interestingly, the average AUC for simultaneous, co-registered OCT and SHG was not significantly different from OCT alone. This suggests that collagen may not be a useful fluorophore for ovarian cancer screening. The high performance of OCT warrants further investigation.

Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.