Timothy J. Muldoon

Subcellular-resolution molecular imaging within living tissue by fiber microendoscopy

Conventional histopathology involves sampling, sectioning and staining of tissue specimens prior to microscopic evaluation, and provides diagnostic information at a single location and point in time. In vivo microscopy and molecular-targeted optical labeling are two rapidly developing fields, which together have the potential to provide anatomical and functional indications of disease by staining and imaging tissue in situ. To address the need for high-resolution imaging instrumentation, we have developed a compact, robust, and inexpensive fiber-optic microendoscopy system based around wide-field LED illumination, a flexible 1 mm diameter fiber-optic bundle, and a color CCD camera. We demonstrate the sub-cellular resolution imaging capabilities of the system through a series of experiments, beginning with simultaneous imaging of three different cancer cell lines in culture, each targeted with a distinct fluorescent label. We used the narrow diameter probe to access subcutaneous tumors in an in vivo murine model, allowing direct comparison of microendoscopy images with macroscopic images and histopathology. A surgically resected tissue specimen from the human oral cavity was imaged across the clinical margin, demonstrating qualitative and quantitative distinction between normal and cancerous tissue based on sub-cellular image features. Finally, the fiber-optic microendoscope was used on topically-stained normal human oral mucosa in vivo, resolving epithelial cell nuclei and membranes in real-time fluorescence images. Our results demonstrate that this imaging system can potentially complement conventional diagnostic techniques, and support efforts to translate emerging molecular-diagnostic and therapeutic agents into clinical use.

High-resolution imaging in Barrett's esophagus: a novel, low-cost endoscopic microscope

BACKGROUND This report describes the clinical evaluation of a novel, low-cost, high-resolution endoscopic microscope for obtaining fluorescent images of the cellular morphology of the epithelium of regions of the esophagus with Barretts metaplasia. This noninvasive point imaging system offers a method for obtaining real-time histologic information during endoscopy. OBJECTIVE The objective of this study was to compare images taken with the fiberoptic endoscopic microscope with standard histopathologic examination. DESIGN Feasibility study. SETTING The University of Texas M.D. Anderson Cancer Center Department of Gastroenterology. PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASUREMENTS: The tissue samples studied in this report were obtained by endoscopic resection from patients with previous diagnoses of either high-grade dysplasia or esophageal adenocarcinoma. RESULTS Three distinct tissue types were observed ex vivo with the endoscopic microscope: normal squamous mucosa, Barretts metaplasia, and high-grade dysplasia. Squamous tissue was identified by bright nuclei surrounded by dark cytoplasm in an ordered pattern. Barretts metaplasia could be identified by large glandular structures with intact nuclear polarity. High-grade dysplasia was visualized as plentiful, irregular glandular structures and loss of nuclear polarity. Standard histopathologic examination of study samples confirmed the results obtained by the endoscopic microscope. LIMITATIONS The endoscopic microscope probe had to be placed into direct contact with tissue. CONCLUSIONS It was feasible to obtain high-resolution histopathologic information using the endoscopic microscope device. Future improvement and integration with widefield endoscopic techniques will aid in improving the sensitivity of detection of dysplasia and early cancer development in the esophagus.

Noninvasive imaging of oral neoplasia with a high-resolution fiber-optic microendoscope.

The purpose of this study was to evaluate the ability of high‐resolution microendoscopy to image and quantify changes in cellular and architectural features seen in early oral neoplasia in vivo.

Molecular imaging of glucose uptake in oral neoplasia following topical application of fluorescently labeled deoxy-glucose

The clinical value of assessing tumor glucose metabolism via F‐18 fluorodeoxyglucose (FDG) PET imaging in oncology is well established; however, the poor spatial resolution of PET is a significant limitation especially for early stage lesions. An alternative technology is optical molecular imaging, which allows for subcellular spatial resolution and can be effectively used with topical contrast agents for imaging epithelial derived cancers. The goal of this study was to evaluate the potential of optical molecular imaging of glucose metabolism to aid in early detection of oral neoplasia. Fluorescently labeled deoxyglucose (2‐NBDG (2‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐2‐deoxy‐D‐glucose)) was applied topically to tissue phantoms, fresh oral biopsies (n = 32) and resected tumors specimens (n = 2). High‐resolution imaging results show that 2‐NBDG can be rapidly delivered to oral epithelium using topical application. In normal epithelium, the uptake of 2‐NBDG is limited to basal epithelial cells. In contrast, high‐grade dysplasia and cancers show uptake of 2‐NBDG in neoplastic cells throughout the lesion. Following 2‐NBDG labeling, the mean fluorescence intensity of neoplastic tissue averages 3.7 times higher than that of matched nonneoplastic oral biopsies in samples from 20 patients. Widefield fluorescence images of 8‐paired oral specimens were obtained pre and postlabeling with 2‐NBDG. Prior to labeling, neoplastic samples showed significantly lower autofluorescence than nonneoplastic samples. The fluorescence of neoplastic samples increased dramatically after labeling; the differential increase in fluorescence was on average 30 times higher in neoplastic samples than in normal samples. Topical application of 2‐NBDG can therefore provide image contrast in both widefield and high‐resolution fluorescence imaging modalities, highlighting its potential in early detection of oral neoplasia.

Evaluation of quantitative image analysis criteria for the high-resolution microendoscopic detection of neoplasia in Barrett's esophagus

Early detection of neoplasia in patients with Barretts esophagus is essential to improve outcomes. The aim of this ex vivo study was to evaluate the ability of high-resolution microendoscopic imaging and quantitative image analysis to identify neoplastic lesions in patients with Barretts esophagus. Nine patients with pathologically confirmed Barretts esophagus underwent endoscopic examination with biopsies or endoscopic mucosal resection. Resected fresh tissue was imaged with fiber bundle microendoscopy; images were analyzed by visual interpretation or by quantitative image analysis to predict whether the imaged sites were non-neoplastic or neoplastic. The best performing pair of quantitative features were chosen based on their ability to correctly classify the data into the two groups. Predictions were compared to the gold standard of histopathology. Subjective analysis of the images by expert clinicians achieved average sensitivity and specificity of 87% and 61%, respectively. The best performing quantitative classification algorithm relied on two image textural features and achieved a sensitivity and specificity of 87% and 85%, respectively. This ex vivo pilot trial demonstrates that quantitative analysis of images obtained with a simple microendoscope system can distinguish neoplasia in Barretts esophagus with good sensitivity and specificity when compared to histopathology and to subjective image interpretation.

Synergistic Photothermal and Antibiotic Killing of Biofilm-Associated Staphylococcus aureus Using Targeted Antibiotic-Loaded Gold Nanoconstructs

Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as “ESKAPE pathogens” on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections.

High-resolution fiber optic microscopy with fluorescent contrast enhancement for the identification of axillary lymph node metastases in breast cancer: a pilot study

This prospective pilot study evaluates the potential of high-resolution fiber optic microscopy (HRFM) to identify lymph node metastases in breast cancer patients. 43 lymph nodes were collected from 14 consenting breast cancer patients. Proflavine dye was topically applied to lymph nodes ex vivo to allow visualization of nuclei. 242 images were collected at 105 sites with confirmed histopathologic diagnosis. Quantitative statistical features were calculated from images, assessed with one-way ANOVA, and were used to develop a classification algorithm with the goal of objectively discriminating between normal and metastatic tissue. A classification algorithm using mean image intensity and skewness achieved sensitivity of 79% (27/34) and specificity of 77% (55/71). This study demonstrates the technical feasibility and diagnostic potential of HRFM with fluorescent contrast in the ex vivo evaluation of lymph nodes from breast cancer patients.

Analysis of skin lesions using laminar optical tomography

Evaluation of suspicious skin lesions by dermatologists is usually accomplished using white light examination and direct punch or surgical biopsy. However, these techniques can be imprecise for estimating a lesion’s margin or level of dermal invasion when planning surgical resection. Laminar optical tomography (LOT) is an imaging technique capable of acquiring depth-sensitive information within scattering tissues. Here, we explore whether LOT data can be used to predict the depth and thickness of pigmented lesions using a range of simulations and phantom models. We then compare these results to LOT data acquired on normal and malignant skin lesions in vivo.

Optical redox ratio identifies metastatic potential-dependent changes in breast cancer cell metabolism.

The development of prognostic indicators of breast cancer metastatic risk could reduce the number of patients receiving chemotherapy for tumors with low metastatic potential. Recent evidence points to a critical role for cell metabolism in driving breast cancer metastasis. Endogenous fluorescence intensity of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) can provide a label-free method for assessing cell metabolism. We report the optical redox ratio of FAD/(FAD + NADH) of four isogenic triple-negative breast cancer cell lines with varying metastatic potential. Under normoxic conditions, the redox ratio increases with increasing metastatic potential (168FARN>4T07>4T1), indicating a shift to more oxidative metabolism in cells capable of metastasis. Reoxygenation following acute hypoxia increased the redox ratio by 43 ± 9% and 33 ± 4% in the 4T1 and 4T07 cells, respectively; in contrast, the redox ratio decreased 14 ± 7% in the non-metastatic 67NR cell line. These results demonstrate that the optical redox ratio is sensitive to the metabolic adaptability of breast cancer cells with high metastatic potential and could potentially be used to measure dynamic functional changes that are indicative of invasive or metastatic potential.

Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation

BACKGROUND Confocal endomicroscopy has revolutionized endoscopy by offering subcellular images of the GI epithelium; however, the field of view is limited. Multiscale endoscopy platforms that use widefield imaging are needed to better direct the placement of high-resolution probes. DESIGN Feasibility study. OBJECTIVE This study evaluated the feasibility of a single agent, proflavine hemisulfate, as a contrast medium during both widefield and high-resolution imaging to characterize the morphologic changes associated with a variety of GI conditions. SETTING The University of Texas MD Anderson Cancer Center, Houston, Texas, and Mount Sinai Medical Center, New York, New York. PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASUREMENTS: Resected specimens were obtained from 15 patients undergoing EMR, esophagectomy, or colectomy. Proflavine hemisulfate, a vital fluorescent dye, was applied topically. The specimens were imaged with a widefield multispectral microscope and a high-resolution microendoscope. The images were compared with histopathologic examination. RESULTS Widefield fluorescence imaging enhanced visualization of morphology, including the presence and spatial distribution of glands, glandular distortion, atrophy, and crowding. High-resolution imaging of widefield abnormal areas revealed that neoplastic progression corresponded to glandular heterogeneity and nuclear crowding in dysplasia, with glandular effacement in carcinoma. These widefield and high-resolution image features correlated well with the histopathologic features. LIMITATIONS This imaging approach must be validated in vivo with a larger sample size. CONCLUSIONS Multiscale proflavine-enhanced fluorescence imaging can delineate epithelial changes in a variety of GI conditions. Distorted glandular features seen with widefield imaging could serve as a critical bridge to high-resolution probe placement. An endoscopic platform combining the two modalities with a single vital dye may facilitate point-of-care decision making by providing real-time, in vivo diagnoses.