This PDF file contains the front matter associated with SPIE Proceedings Volume 6432, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Confocal and multi-photon imaging systems are currently miniaturized to fit them to endoscopic size requirements of probe diameters often less than 2.8 mm. High resolution in lateral dimensions of less than 1 micron and in axial dimensions of less than 10 microns is desired to resolve sub-cellular details of in-vivo tissue. GRIN rod lens systems have been widely used as high numerical aperture objectives in these applications because of their small size, good image quality and favourable geometry with plane optical surfaces, which allows easy assembly. Image generation has been enabled by combination with coherent imaging fiber bundles, GRIN relay lenses and scanning single fibers or photonic band gap fibers. With previous GRIN systems of maximum NA of 0.50, a resolution limit laterally of approx. 1.0 micron and axially of 5.5 to 10 microns was obtained in the case of two-photon excitation. Here, we introduce a novel concept of a GRIN lens objective system with significantly higher NA yielding resolution improved by a factor of two (lateral) and four (axial). The image quality of initial 1.0 mm GRIN components will be characterized by the signal analysis of 0.2 micron fluorescent beads in the multi-photon scheme and by conventional image tests using test grids with transmitting illumination. Potentials for further miniaturization and for changing the direction of view will be discussed.

We present a modified multi-spectral configuration of a slit-scanning confocal microendoscope that provides higher spectral resolution in a fully automated interface. Tissue fluorescence signal is directed through a dispersive element that spreads the spectral information across the CCD camera mapping spectral information perpendicular to the confocal slit. The dispersive element may be chosen to meet the specific requirements defined by the user. Our current system uses a BK7 prism with a 45^o wedge angle and a 20mm diameter clear aperture. The prism is shifted from the optical axis allowing automated switching from grayscale (beam on-axis) to multi-spectral (beam off-axis) imaging by tilting a computer controlled mirror. The system records over a spectral range of 450nm to 750nm. The minimum resolvable wavelength difference varies from 2.1nm to 8.3nm over the spectral range. The lateral and axial resolution of the system is approximately 3&mgr;m by 30&mgr;m, respectively, and is the same for both grayscale and multi-spectral imaging modes. Multi-spectral imaging results from ex-vivo tissues are presented.

Purpose: Optical coherence tomography (OCT) is a minimally invasive, depth-resolved imaging tool that can be commissioned for small diameter endoscopic applications for imaging mouse models of colorectal cancer. In this study, we utilized ultrahigh resolution OCT (UHR OCT) to serially image the lower colon of azoxymethane (AOM) treated A/J mouse models of CRC, monitor the progression of neoplastic transformations, and determine if OCT is capable of identifying early disease. Experimental Design: Thirteen AOM treated A/J and two control A/J mice were surveyed at four timepoints (8, 14, 22, and 26 weeks post AOM treatment) using a prototype 2.0 mm diameter UHR OCT endoscope-based system that achieved resolutions of 3.2 um axial and 4.4 um lateral. Histological samples were obtained at the final imaging timepoint serving as the gold standard. Results: Gross and histological assessment of the excised colonic tissue revealed at least one tumor in all 13 AOM treated mice, with most mice developing multiple tumors. In the corresponding OCT images, a progression from healthy thin mucosa to adenoma appearing as large, structurally disorganized masses was visualized over the imaging time points correlating to the locations of the grossly visualized tumors. Conclusions: This study indicates that UHR OCT enables accurate identification of disease and non-destructive visualization of CRC progression in the lower colon of mice.

Statistical analysis of endoscopic optical coherence tomography (EOCT) surveillance of 78 patients with Barrett's esophagus (BE) is presented in this study. The sensitivity of OCT device in retrospective open detection of early malignancy (including high grade dysplasia and intramucosal adenocarcinoma (IMAC)) was 75%, specificity 82%, diagnostic accuracy - 80%, positive predictive value- 60%, negative predictive value- 87%. In the open recognition of IMAC sensitivity was 81% and specificity were 85% each. Results of a blind recognition with the same material were similar: sensitivity - 77%, specificity 85%, diagnostic accuracy - 82%, positive predictive value- 70%, negative predictive value- 87%. As the endoscopic detection of early malignancy is problematic, OCT holds great promise in enhancing the diagnostic capability of clinical GI endoscopy.

A 5mm biophotonic catheter was conceived for optical coherence tomography (OCT) with collimation optics, an axicon lens, and custom design imaging optics, yielding a 360 degree scan aimed at imaging within concave structures such as lung lobes. In OCT a large depth of focus is necessary to image a thick sample with a constant high transverse resolution. There are two approaches to achieving constant lateral resolution in OCT: Dynamic focusing or Bessel beam forming. This paper focuses on imaging with Bessel beams. A Bessel beam can be generated in the sample arm of the OCT interferometer when axicon optics is employed instead of a conventional focusing lens. We present a design for a 5mm catheter that combines an axicon lens with imaging optics and the coupling of a MEMS mirror attached to a micromotor that allow 360 degree scanning with a resolution of about 5 microns across a depth of focus of about 1.2mm.

Design and development of a 3D scanning MEMS micromirror integrated miniaturized optical probe has been presented in this article. The probe is designed to be less than 2 mm in diameter and has dynamic scanning modality for larger field of view. Scanning is achieved using 3D micromirror device, which has 16º out of plane and 360º beam rotation capability. Initial target of 45º out of plane deflection is yet to be achieved. The probe being developed currently would have scanning capability in one quarter of 360º full rotation. The field of view would still be very large and multiple optical biopsies would be possible for planned cancer model diagnostics. The feasibility of using scanning mirror into an optical probe was demonstrated using scanning repeatability and OCT imaging tests. Geometrical optics and package design using silicon optical bench have been established. Miniaturized 3D scanning micromirror have been designed and developed with 16º out of plane deflection demonstrated. Probe package integration and optical testing are carried out.

Confocal fluorescence high resolution imaging during standard endoscopic procedures has been presented as a very promising tool to enhance patient care and physician practice by providing supplementary diagnostic information in real-time. The purpose of this paper is to show not only potential, but convincing results of endoscopic microscopy using a catheter-based approach. Mauna Kea Technologies' core technology, Cellvizio, delivers dynamic imaging at 12 frames/second using confocal miniprobes inserted through the operating channel of regular endoscopes. Cellvizio is composed of 3 parts including (a) a Laser Scanning Unit, (b) Confocal Miniprobe^TM with the following characteristics: 5-15 &mgr;m axial resolution, 2-5 &mgr;m lateral resolution, 15-100 &mgr;m depth of penetration, field of view of 600x500 &mgr;m and (c) a software package with onthe- fly processing capabilities. With several tens of patients examined during routine GI endoscopy procedures, the most relevant clinical parameters could be assessed in a doubled-blinded fashion between the endoscopist and a pathologist and results showing very high accuracy in the differentiation of neoplasia from normal and hyperplastic tissue were obtained. In the field of pulmonology, the micro-autofluorescence properties of tissues could be assessed and structures never before accessed in vivo were observed. Cellvizio® may be useful to study bronchial remodeling in asthma and chronic obstructive pulmonary diseases. Using appropriate topical fluorescent dye, the Confocal Miniprobes may also make it possible to perform optical biopsy of precancerous and superficial bronchial cancers. Cellvizio® is as a new tool towards "targeted biopsies", leading to earlier, more reliable and cost effective diagnostic procedures. Other applications, specifically in molecular imaging are also made possible by the miniaturization of the probe (combination with biopsy needle for solid organs use or lymph node detection) and by the compatibility of the system with other imaging modalities (auto-fluorescence and narrow-band imaging endoscopy, MRI, PET, etc).

In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of <1mm diameter to transfer the confocal imaging plane to tissue in intact small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and sub-cellular details could be readily visualised in vivo at high resolution. In rodent disease models, in vivo endomicroscopy with appropriate fluorescent agents allowed examination of thrombosis formation, tumour microvasculature and liver metastases, diagnosis and staging of ulcerative colitis, liver necrosis and glomerulonephritis. Miniaturised confocal endomicroscopy allows rapid in vivo molecular and subsurface microscopy of normal and pathologic tissue at high resolution in small and large whole animal models. Fluorescein endomicroscopy has recently been introduced into the medical device market as a clinical imaging tool in GI endoscopy and is undergoing clinical evaluation in laparoscopic surgery. This medical usage is encouraging in-situ endomicroscopy as an important pre-clinical research tool to observe microscopic and molecular system biologic events in vivo in animal models for various human diseases.

A mobile confocal microendoscope for use in a clinical setting has been developed. This system employs an endoscope consisting of a custom designed objective lens with a fiber optic imaging bundle to collect in-vivo images of patients. Some highlights and features of this mobile system include frame rates of up to 30 frames per second, an automated focus mechanism, automated dye delivery, clinician control, and the ability to be used in an area where there is a single 110V outlet. All optics are self-contained and the entire enclosure and catheter can be moved between surgical suites, sterilized and brought online in under 15 minutes. At this time, all data have been collected with a 488 nm laser, but the system is able to have a second laser line added to provide additional imaging capability. Preliminary in vivo results of images from the ovaries using topical fluorescein as a contrast agent are shown. Future plans for the system include use of acridine orange (AO) or SYTO-16 as a nucleic acid stain.

Colorectal cancer is the second leading cause of cancer-related death in the United States. Approximately 50% of these deaths could be prevented by earlier detection through screening. Magnification chromoendoscopy is a technique which utilizes tissue stains applied to the gastrointestinal mucosa and high-magnification endoscopy to better visualize and characterize lesions. Prior studies have shown that shapes of colonic crypts change with disease and show characteristic patterns. Current methods for assessing colonic crypt patterns are somewhat subjective and not standardized. Computerized algorithms could be used to standardize colonic crypt pattern assessment. We have imaged resected colonic mucosa in vitro (N = 70) using methylene blue dye and a surgical microscope to approximately simulate in vivo imaging with magnification chromoendoscopy. We have developed a method of computerized processing to analyze the crypt patterns in the images. The quantitative image analysis consists of three steps. First, the crypts within the region of interest of colonic tissue are semi-automatically segmented using watershed morphological processing. Second, crypt size and shape parameters are extracted from the segmented crypts. Third, each sample is assigned to a category according to the Kudo criteria. The computerized classification is validated by comparison with human classification using the Kudo classification criteria. The computerized colonic crypt pattern analysis algorithm will enable a study of in vivo magnification chromoendoscopy of colonic crypt pattern correlated with risk of colorectal cancer. This study will assess the feasibility of screening and surveillance of the colon using magnification chromoendoscopy.

We have developed the novel video endoscope imaging techniques; Narrow band imaging (NBI), Auto-Fluorescence Imaging (AFI), Infra-Red Imaging (IRI) and Endo-Cytoscopy System (ECS). The purpose of these imaging techniques is to emphasize the important tissue features associated with early stage of lesions. We have already launched the new medical endoscope system including NBI, AFI and IRI (EVIS LUCERA SPECTRUM, OLYMPUS MEDICAL SYSTEMS Co., Ltd., Fig.1). Moreover ECS, which has enough magnification to observe cell nuclei on a superficial mucosa under methylene blue dye staining, is the endoscopic instrument with ultra-high optical zoom. In this paper we demonstrate the concepts and the medical efficacy of each technology.

The technique of Fluorescence Excitation Ratioometry is presented and the limits of its precision are discussed. Further applications of the Flicker Fluorimetry technique to polarimetry, colorimetry and fluorescence emission spectroscopy are described with measurements and calibrations presented.

Micromirrors used to steer optical beams at the tip of an endoscope are an active area of research. MEMS mirrors with various actuation mechanisms have been reported including rotating micromotors, parallel plate electrostatic drive, electrothermal and electrostatic comb drive. This paper presents a two-axis magnetically actuated micromirror designed for an OCT (Optical Coherence Tomography) endoscope. Magnetic actuation allows large angular deflections at low voltage (1-3 V), a significant advantage for patient safety relative to the high voltages used for electrostatic mirrors. Actuation is accomplished with small coils in proximity to the moving mirror, which contains a small permanent magnet. The endoscope scan engine is contained in a 2.8 mm ID plastic tube. The MEMS scan mirror was fabricated by a simple process using an SOI wafer and only 2 photo-steps. The mirror is supported by silicon springs on both axes, and can scan to +/- 20 degrees mechanical on both axes. Both time domain and spectral domain OCT have been used to take cross-sectional tissue images. By operating the 2-axis mirror in a raster scan, a sequence of cross-sections is taken to form a 3-D image. Details of the endoscope design, MEMS fabrication, and sample OCT images are presented.