Use of in vivo near-infrared laser confocal endomicroscopy with indocyanine green to detect the boundary of infiltrative tumor.

OBJECT: Infiltrative tumor resection is based on regional (macroscopic) imaging identification of tumorous tissue and the attempt to delineate invasive tumor margins in macroscopically normal-appearing tissue, while preserving normal brain tissue. The authors tested miniaturized confocal fiberoptic endomicroscopy by using a near-infrared (NIR) imaging system with indocyanine green (ICG) as an in vivo tool to identify infiltrating glioblastoma cells and tumor margins. METHODS: Thirty mice underwent craniectomy and imaging in vivo 14 days after implantation with GL261-luc cells. A 0.4 mg/kg injection of ICG was administered intravenously. The NIR images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope probe. Histological samples were acquired from matching imaged areas for correlation of tissue images. RESULTS: In vivo NIR wavelength confocal endomicroscopy with ICG detects fluorescence of tumor cells. The NIR and ICG macroscopic imaging performed using a surgical microscope correlated generally to tumor and peritumor regions, but NIR confocal endomicroscopy performed using ICG revealed individual tumor cells and satellites within peritumoral tissue; a definitive tumor border; and striking fluorescent microvascular, cellular, and subcellular structures (for example, mitoses, nuclei) in various tumor regions correlating with standard clinical histological features and known tissue architecture. CONCLUSIONS: Macroscopic fluorescence was effective for gross tumor detection, but NIR confocal endomicroscopy performed using ICG enhanced sensitivity of tumor detection, providing real-time true microscopic histological information precisely related to the site of imaging. This first-time use of such NIR technology to detect cancer suggests that combined macroscopic and microscopic in vivo ICG imaging could allow interactive identification of microscopic tumor cell infiltration into the brain, substantially improving intraoperative decisions.

Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model.

INTRODUCTION: Recent developments in optical science and image processing have miniaturized the components required for confocal microscopy. Clinical confocal imaging applications have emerged, including assessment of colonic mucosal dysplasia during colonoscopy. We present our initial experience with handheld, miniaturized confocal imaging in a murine brain tumor model. METHODS: Twelve C57/BL6 mice were implanted intracranially with 10(5) GL261 glioblastoma cells. The brains of 6 anesthetized mice each at 14 and 21 days after implantation were exposed surgically, and the brain surface was imaged using a handheld confocal probe affixed to a stereotactic frame. The probe was moved systematically over regions of normal and tumor-containing tissue. Intravenous fluorescein and topical acriflavine contrast agents were used. Biopsies were obtained at each imaging site beneath the probe and assessed histologically. Mice were killed after imaging. RESULTS: Handheld confocal imaging produced exquisite images, well-correlated with corresponding histologic sections, of cellular shape and tissue architecture in murine brain infiltrated by glial neoplasm. Reproducible patterns of cortical vasculature, as well as normal gray and white matter, were identified. Imaging effectively distinguished between tumor and nontumor tissue, including infiltrative tumor margins. Margins were easily identified by observers without prior neuropathology training after minimum experience with the technology. CONCLUSION: Miniaturized handheld confocal imaging may assist neurosurgeons in detecting infiltrative brain tumor margins during surgery. It may help to avoid sampling error during biopsy of heterogeneous glial neoplasms, with the potential to supplement conventional intraoperative frozen section pathology. Clinical trials are warranted on the basis of these promising initial results.

A pilot study of the feasibility of confocal endomicroscopy for examination of the human airway.

BACKGROUND: Traditional methods of evaluating human airway histology, such as surgical biopsy or endobronchial biopsy, are limited by the risks associated with these tissue-sampling procedures. OBJECTIVE: The purpose of this study was to develop and evaluate the first confocal endomicroscope for real-time, in vivo imaging of human respiratory mucosa in a clinical setting. METHODS: A confocal endomicroscope prototype was designed using Pentax bronchoscope parts (EB1970K). Airways of adult patients (N=5) undergoing rigid bronchoscopy for various clinical indications were imaged with the confocal endomicroscope after intravenous administration of fluorescein sodium. The device was introduced into the airways through the rigid bronchoscope. Images were collected from the trachea, primary and secondary carinae, and any endobronchial mass. The images were compared with those obtained from histologic sections from conventional endobronchial biopsies. RESULTS: Confocal endomicroscopy provided real-time images of the cellular and subcellular structures of the respiratory mucosa and submucosa in vivo. The pseudostratified columnar epithelium (including columnar cells and goblet cells) could be visualized. Images obtained at increasing depth showed the lamina propria and microvasculature. Longitudinal folds in the mucosa enabled imaging in cross-section, showing alignment of epithelial cells along the basement membrane and cilia on the surface of the cells. Below the epithelium, the smooth muscle could be identified. In images from a patient with an endobronchial adenocarcinoma, confocal imaging could distinguish between a normal airway epithelium and malignant tissue. CONCLUSIONS: Confocal endomicroscopy is a feasible method for analyzing human airway wall architecture and endobronchial abnormalities in histologic detail in vivo.

In vivo confocal endomicroscopy in the diagnosis and evaluation of celiac disease.

BACKGROUND & AIMS: Accurate histopathology of endoscopic duodenal biopsy specimens is critical in the diagnosis of celiac disease (CD) but sampling error and poor quality specimens may generate a false-negative result. Confocal endomicroscopy (CEM) is a novel technology allowing real-time in vivo microscopy of the mucosa that may diagnose CD and evaluate its severity and response to treatment more accurately than histopathology. METHODS: Subjects with CD and controls prospectively underwent CEM. Features of villous atrophy and crypt hypertrophy were defined. A CEM score measuring CD severity was devised and validated against the diagnosis of CD and blinded histopathology. Receiver operator characteristics, sensitivity to change after treatment, and reliability of findings were assessed. RESULTS: From 31 patients (6 untreated CD, 11 treated CD, and 14 controls), 7019 CEM images paired with 326 biopsy specimens were obtained. The accuracy of CEM in diagnosing CD was excellent (receiver operator characteristics area under the curve, 0.946; sensitivity, 94%, specificity, 92%) and correlated well with the Marsh grading (R-squared, 0.756). CEM differentiated CD from controls (P < .0001) and was sensitive to change after treatment with gluten-free diet (1787 optical biopsies; P = .012). The intraclass correlation of reliability was high (0.759-0.916). Of the 17 cases with diagnosed CD, 16 (94%) were diagnosed correctly using CEM but only 13 (76%) had detectable histopathology changes. The procedure was safe and well-tolerated. CONCLUSIONS: CEM effectively diagnoses and evaluates CD severity in vivo. This promising technique has the potential to improve endoscopy efficiency.

Pentax confocal endomicroscope: a novel imaging device for in vivo histology of the upper and lower gastrointestinal tract.

With advances in endoscopic imaging tools, it is becoming increasingly possible to find, assess and treat numerous gastrointestinal (GI) pathologies at the time of endoscopy. This article focuses on the newly developed Pentax confocal endomicroscope that enables in vivo microscopic imaging of the upper and lower GI tract in histological detail at the time of patient examination without the collection of biopsies. The optical imaging technique has the potential to revolutionize clinical workflows in endoscopy through high-resolution fluorescence imaging of cellular and subcellular detail from the surface and subepithelial layers of the GI mucosa. The device incorporates a full screen, high-resolution charge coupled device as well as a confocal microscope. Preliminary data from blinded clinical studies suggests that the use of this device can increase the diagnostic yield for disease detection in conditions such as ulcerative colitis and Barrett's esophagus, where diagnosis using white light endoscopy is problematic. The early detection and diagnosis of GI abnormalities through the collection of 'optical' biopsies or targeted mucosal excisional biopsies has the potential to improve patient outcomes and enable early therapeutic intervention.

A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract.

BACKGROUND: This report describes the development and the clinical evaluation of a novel confocal endomicroscope for obtaining fluorescence images of cellular morphology of the mucosae of the upper- and the lower-GI tract in vivo. The work assessed the feasibility of performing in vivo microscopy at endoscopic examination and evaluated fluorescence imaging protocols. METHODS: Images were collected in real time by using two prototype endoscope configurations, featuring slightly different miniaturized fiber-optic confocal microscopes, fitted integrally into the tips of conventional endoscopes. Confocal scanning was performed at 488 nm illumination for excitation of exogenously applied fluorophores (topical acriflavine and intravenous fluorescein). The images were compared with conventional histology of biopsy specimens and the findings of white-light endoscopy. RESULTS: Confocal endomicroscopy enabled imaging of cellular and subcellular structures (i.e., nuclei) of the GI tract. The crypts of the colonic mucosa, the villi of the terminal ileum and duodenum, the gastric pits of the stomach, and the squamous epithelium of the distal esophagus could be clearly visualized. Acriflavine strongly contrasted the cell nuclei of the surface epithelium, including the absorptive epithelial cells and the mucous secreting goblet cells. Fluorescein stained the extracellular matrix of the surface epithelium and also the subepithelial layers of the lamina propria. Images at increasing depth beneath the epithelium showed the mucosal capillary network. The findings correlated with the histology of biopsy specimens. CONCLUSIONS: The development of a fluorescence confocal endomicroscope makes it practical to examine the upper- and the lower-GI mucosa in cellular detail during otherwise routine endoscopic examination. The results represent a major technical advance in the development of this new optical imaging modality for the in vivo examination of GI tissue.

View of normal human skin in vivo as observed using fluorescent fiber-optic confocal microscopic imaging.

Fluorescence confocal scanning laser microscopy, using a miniaturized handheld scanner, was performed to visualize the microscopic architecture of normal human epidermis in vivo. Fluorescein sodium (approximately 20 microL of 0.2% wt/vol) was administered via intradermal injection to normal skin on the volar forearm of 22 patients. The skin was imaged continuously from 1 to 15 min after injection. Fluorescein was excited at 488 nm and the fluorescent emission was detected at > 505 nm. In each subject, a series of images was collected at increasing depth, from superficial stratum corneum to papillary dermis. Features observed in confocal images were compared to those seen in hematoxylin- and eosin-stained sections of skin. The confocal images demonstrated the architecture of superficial skin in the horizontal plane. There was a transition in keratinocyte size, shape, and morphology with progressive imaging into the deeper epidermal layers. Superficial dermis and microscopic capillaries with blood flow were easily observed. The morphologic patterns associated with the major cell types of the epidermis were consistent with those known from conventional histology. We report the ability of in vivo fluorescence point scanning laser confocal microscopy to produce real-time, high-resolution images of the microscopic architecture of normal human epidermis using a noninvasive imaging technology.

In vivo detection of morphological and microvascular changes of the colon in association with colitis using fiberoptic confocal imaging (FOCI).

Using a well-established rodent model of inflammatory bowel disease (IBD), the present study examined changes in the microvasculature of the colonic mucosa in association with ulcerative colitis (UC). The results were compared to microscopic alterations in tissue morphology to establish a temporal relationship between microcirculatory dysfunction and IBD pathology. Mild colitis was induced in rats by the oral consumption of 5% dextran sulfate sodium (DSS) in drinking water. Control animals were provided with water ad libitum. After 3, 5, and 7 days of oral ingestion of DSS, anesthetized rats were laparotomized. The mucosal surface of the distal colon was then examined using fiber optic confocal imaging (FOCI; excitation 488 nm argon ion laser, detection above 515 nm). Changes in the mucosal architecture were examined following the topical application of the fluorescent dye, tetracycline hydrochloride. Tetracycline hydrochloride, an antibiotic used widely in clinical medicine, enabled imaging of the crypts at the surface of the mucosa. Spatial changes in the microvascular structure were assessed following the intravenous administration of fluorescein isothiocyanate dextran (FITC-dextran). Confocal images were correlated with clinical parameters, including weight loss, occult blood, and stool consistency. Attenuation of the colonic epithelium was detected on day 3 colitis. Morphological changes including crypt loss, crypt distortion, and inflammatory cell infiltrate were detected on day 5 and day 7 colitis. Dual channel imaging showed the mucosal capillary network outlining the stromal confines of the mucus-secreting glands in control tissue. Experimental colitis resulted in diffuse hypervascularity and tortuosity of the capillary vessels. Evidence of increased vessel leakiness (leakage of FITC-dextran from the lumen) was first detected on day 5 colitis. Complete disruption of the normal honeycomb pattern of the vessels and capillary dilation was evident after 7 days of DSS ingestion. These findings suggest that the pathogenesis of ulcerative colitis is associated with changes in the vascular architecture as demonstrated in vivo using confocal microscopy.