This PDF file contains the front matter associated with SPIE Proceedings Volume 6851, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We have developed a mobile confocal microendoscope system that provides live cellular imaging during surgery to aid in diagnosing microscopic abnormalities including cancer. We present initial clinical trial results using the device to image ovaries in-vivo using fluorescein and ex-vivo results using acridine orange. The imaging catheter has improved depth control and localized dye delivery mechanisms than previously presented. A manual control now provides a simple way for the surgeon to adjust and optimize imaging depth during the procedure while a tiny piezo valve in the imaging catheter controls the dye delivery.

Fibered confocal fluorescence microscopy (FCFM) is an emerging technique that can be used during bronchoscopy to analyze the nature of the human bronchial mucosa and alveolar network fluorescence microstructure. An endoscopic fibered confocal fluorescence microscopy system with spectroscopic analysis capability was developed allowing realtime, simultaneous images and emission spectra acquisition, at 488 nm excitation, using a flexible miniprobe. This flexible 1.4 mm miniprobe can be introduced into the working channel of a flexible endoscope and gently advanced through the bronchial tree up to the alveoli. FCFM in conjunction with bronchoscopy is able to image the in vivo autofluorescence microstructure of the bronchial mucosa but also the alveolar respiratory network outside of the usual field of view. In the normal bronchi, reproducible images were obtained, characterized by a highly organized fibered network. Precancerous lesions exhibited alterations of this fibered network. Microscopic and spectral analysis showed that the signal mainly originates from the elastin component of the bronchial subepithelial layer. In non smokers, the system images the elastin backbone of the aveoli. In active smokers, a strong autofluorescence signal appears from alveolar macrophages. The FCFM technique appears promising for in vivo exploration of the bronchial and alveolar extracellular matrix.

Ovarian cancer is the fourth leading cause of cancer-related death among women in the United States. If diagnosed at an early stage, the 5-year survival rate is 94%, but drops to 68% for regional disease and 29% for distant metastasis; only 19% of all cases are diagnosed at the early, localized stage. Optical coherence tomography is a recently emerging non-destructive imaging technology, achieving high axial resolutions (10-20 microns) at imaging depths up to 2 mm. Previously, we studied OCT imaging in normal and diseased human ovary ex vivo to determine the features OCT is capable of resolving. Changes in collagen were suggested with several of the images that correlated with changes in collagen seen in malignancy. Areas of necrosis and blood vessels were also visualized using OCT, indicative of an underlying tissue abnormality. We recently developed a custom side-firing laparoscopic OCT (LOCT) probe fabricated specifically for in vivo laparoscopic imaging. The LOCT probe consists of a 38 mm diameter handpiece terminated in an 280 mm long, 4.6 mm diameter tip for insertion into the laparoscopic trocar and is capable of obtaining up to 9.5 mm image lengths at 10 micron axial resolution. In this study, we utilize the LOCT probe to image one or both ovaries of 20 patients undergoing laparotomy or transabdominal endoscopy and oophorectomy to determine if OCT is capable of identifying and/or differentiating normal and neoplastic ovary. To date, we have laparoscopically imaged the ovaries of ten patients successfully with no known complications.

Optical coherence tomography, optical coherence microscopy, reflectance confocal microscopy, and darkfield microscopy all derive contrast from the intensity of endogenous tissue scatter. We have imaged excised mouse colon tissue with these complimentary technologies to make conclusions about structural origins of scatter in the mouse colonic mucosa observed with endoscopic OCT. We find hyperintense scattering both from the cytoplasm of epithelial cells and from the boundary between epithelia and the lamina propria. We find almost no scatter from the portion of epithelial cells containing the nucleus. These observations substantiate explanations for the appearance of colonic crypts and the luminal surface.

Two-photon autofluorescence imaging offers the analysis of cells and tissues without the need of taking biopsies, staining and complicated confocal detection systems. Therefore, it is of special interest for non- or minimal invasive clinical diagnostics. Until now, two-photon imaging was performed only on superficial surfaces like skin or of biopsies. To extend this technique to deeper tissues or inside the body the optical properties have to be reduced to endoscopical sizes. This can be achieved by tiny GRIN-optics, based on a radial gradient in the reflective index. A newly developed GRIN-lens assembly with increased numerical aperture is of special interest which is shown by the quality of tissue constituents and cell autofluorescence images. A fiber directs the laser light to the specimen in an assembly like an endoscope. This well-characterized photonic crystal fiber supports the high laser power of the femtosecond excitation impulses without the generation of non-linearities. A sensitive PMT detector detects the fluorescence. First fluorescence images using a fiber-GRIN lens combination were taken.

The practical limitation of in vivo multiphoton excitation microscopy (MPM) is the lack of a compact and flexible probe. Most MPM depends on the bench-top microscope, which prohibits expansion of in vivo application. In this study, we introduced a miniaturized MPM probe using a microelectromechanical system (MEMS) scanning mirror and a doubleclad photonic crystal fiber (DCPCF). Benefits from both a MEMS mirror and a DCPCF overcome current obstacles for probe development, such as size reduction, rapid scanning, efficient delivery of short pulses, and high collection rate of fluorescent signals. In this study, the completed probe was 1 cm in outer diameter and 14 cm in length. The probe was then integrated to the MPM system and used to image fluorescent beads, paper and biological specimens.

In recent years there has been growing interest in using confocal microscopy to observe tissue structure and function for in vivo pathology. Although confocal microscopy can provide image resolution that is comparable to histopathology, it can be limited by a small field-of-view as well as a low signal-to-noise ratio. In this paper we show that image mosaicing can enhance confocal microscopy by stitching multiple images together to widen the field-of-view and increase the signal-to-noise ratio. Specifically, we present a real-time image mosaicing system for imaging human skin with a hand-held dual-axes confocal microscope. Our system allows the user to "paint" an image mosaic in real-time and aids navigation by localizing the current view with respect to the larger image map. We first discuss image calibration, then describe an efficient algorithm for real-time image mosaicing, and finally present experimental results obtained in vivo with a dual-axes confocal microscope.

This paper reports the study of a Swept Source Optical Coherence Tomography (SS-OCT) setup to remove depth degeneracy for measurements performed with a rotating probe. One of the main drawbacks of SS-OCT is its inability to differentiate positive and negative depths. Some setups have already been proposed to remove this depth ambiguity by introducing a modulation by means of electro-optic or acousto-optic modulators. For cross-sectional imaging, we developed a setup that uses a piezoelectric fiber stretcher to generate a periodic phase shift between successive A-scans, thus introducing a transverse modulation. The depth ambiguity is then resolved by performing a Fourier processing in the transverse direction before processing the data in the axial direction. This approach can also be applied to a rotating probe with a cylindrical geometry by introducing phase shifts between A-scans belonging to successive rotations or between successive B-scans. In the later case, the depth degeneracy is removed by first performing a Fourier processing along the cylindrical axis. We validate this approach by processing images acquired with our catheterized probe based on a rotating fiber and fitted with a GRIN lens and a prism at the tip.

Several technical problems have to be overcome before Optical Coherence Tomography (OCT) can be accepted among the established endoscopic imaging modalities. Most of conventional Michelson-based OCT systems need to have two separated paths of the sample and reference arms, which limits the flexibility of endoscopic probe. Recently, common-path interferometer based OCT have been demonstrated to circumvent the mismatch problems of length, polarization, and dispersion between the reference and sample arms, but the interferometric scanning methods have been realized with time-domain PZT or spectral-domain CCD. In this work, we demonstrate a novel Fourier-domain common-path OCT based on sweeping laser source, which shows superiority in the speed and robustness. Using a holey optical fiber with low bending loss, a novel curled optical patch cord, like a curl cord of telephone, is also adapted for the convenient access to the biological target at the flexible distance. The freedom to use an arbitrary length and wiring of the probe can provide more flexibility for use in endoscopic OCT.

Endoscopic techniques are commonly used for esophageal and gastrointestinal screening. In this process, atypical regions are identified by gross visual and morphological changes. These regions are then biopsied for pathological confirmation prior to determining treatment. In an effort to increase the sensitivity of endoscopic screening, many groups have performed work in developing microscopic endoscopes capable of inspecting tissues on a cellular level. These microscopic endoscopes are generally implemented as either a stand-alone fiber or through the working channel of a traditional endoscope, and are oriented in a manner similar to traditional flexible endoscopes, imaging the region directly ahead of the endoscope with a wide-angle lens. However, this may not be the optimum configuration for microscopic inspection of luminal sidewalls. We present a novel optical configuration for an endoscope that can simultaneously function as a traditional forward-viewing macroscopic endoscope and as a sidewall-viewing microscopic endoscope. With the first prototype, we have realized a water-emersion microscopic that is capable of imaging tissues on a single-cell level. In addition, microscopic side-port configuration enables efficient mapping of the luminal wall. Utilizing simultaneous macroscopic and microscopic imaging, we are developing software for image registration and analysis that will enable localization of microscopic features within a macroscopic frame of reference. Through a combination of microscopic sidewall imaging and software for image analysis, we aim to provide the clinician with the equivalent of an in vivo biopsy, increasing screening effectiveness and decreasing discomfort and costs related to performing multiple biopsies of suspected regions.

In previous work we have demonstrated the utility of laser-induced fluorescence (LIF) and optical coherence tomography (OCT) to identify adenoma in mouse models of colorectal cancer with high sensitivity and specificity. However, improved sensitivity to early disease, as well as the ability to distinguish confounders (e.g. fecal contamination, natural variations in mucosal thickness), is desired. In this study, we investigated the signal enhancement of fluorescent and scattering contrast agents in the colons of AOM-treated mice. The fluorescent tracer scVEGF/Cy, targeted to receptors for vascular endothelial growth factor, was visualized on a dual modality OCT/LIF endoscopic system with 1300-nm center wavelength OCT source and 635-nm LIF excitation. Scattering agents were tested with an 890-nm center wavelength endoscopic OCT system. Agents included nanoshells, 120-nm in diameter, and nanorods, 20-nm in diameter by 80-nm in length. Following imaging, colons were excised. Tissue treated with fluorophore was imaged on an epifluorescence microscope. Histological sections were obtained and stained with H&E and silver enhancer to verify disease and identify regions of gold uptake, respectively. Non-specific signal enhancement was observed with the scattering contrast agents. Specificity for adenoma was seen with the scVEGF/Cy dye.

We present a novel fluorescence sectioning endoscopy that is based on a fiber-bundle and using speckle pattern illumination. The laser speckle pattern is produced by a rotating diffuser and delivered into the fiber bundle for whole-field excitation of the sample. The fluorescence is collected and transmitted to a CCD camera via the endoscope optics and fiber bundle. A sequence of fluorescent images are acquired and processed to reconstruct a 2D depth-resolved image of the sample. The fiber-bundle based endoscopy has similar sectioning capability to that of a laser scanning confocal microscopy but without scanning. Its other advantages include compactness and low cost, which makes it potentially viable for implementation in a portable clinical system.