This PDF file contains the front matter associated with SPIE Proceedings Volume 8575, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology. The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

In order to diagnose cancer in breast tissue, a sample must be removed, prepared, and examined under a microscope. To provide an alternative to conventional biopsies, an endomicroscope intended to perform optical biopsies is demonstrated. The system provides high resolution, high contrast images in real-time which could allow a diagnosis to be made during surgery without the need for tissue removal. Optical sectioning is achieved via structured illumination to reject out of focus light. An image is relayed between the sample plane and the imaging system by a coherent fiber bundle with an achromatized objective lens at the distal tip of the fiber bundle which is the diameter of a biopsy needle. The custom, plastic objective provides correction for both the excitation and emission wavelengths of proflavine (452 nm and 515 nm, respectively). It also magnifies the object onto the distal tip of the fiber bundle to increase lateral resolution. The lenses are composed of the optical plastics Zeonex E48R, PMMA, and polystyrene. The lenses are fabricated via single point diamond turning and assembled using a zero alignment technique. The lateral resolution and chromatic focal shift were measured and in vitro images of breast carcinoma cells stained with proflavine were captured. The optical biopsy system is able to achieve optical sectioning and to resolve smaller features than the current high resolution microendoscope.

We have developed a near infrared (NIR) angioscope that takes multi-wavelength images in 1.7μm band for visualizing lipid-rich coronary plaques. The angioscope comprises light source, camera, and angioscopic catheter. The light source, containing a supercontinuum source and a switching optical filter, emits 1.60, 1.65, 1.73 and 1.76μm wavelengths sequentially in synchronization to the camera frame. The supercontinuum is seeded by 1.55μm wavelength pulses, whose spectrum is spread by an optical fiber with ring loops for reducing peak power so that light in 1.7μm band is generated efficiently. The switching filter contains 1×4 fiber-optic path switches and interferometric band-pass filters. The camera detects NIR images by an InGaAs/GaAsSb type-II quantum well sensor at 100 frames/s. The source wavelength and the camera frame are synchronized with each other by an FPGA. The angioscopic catheter, based on a silica-based image-guide designed for 1.7 μm wavelength, transmits 1300-pixel NIR images and has 0.73 mm outer diameter, which is compatible with the conventional angioscope and suited for continuous flushing to displace blood. We have also developed image processing software that calculates spectral contribution of lipid as lipid score at each pixel and create lipid-enhanced color images at 12 frames/s. The system also includes conventional visible light source and camera, and takes visible light images synchronously with the lipid-enhanced images. The performance of the angioscope for detecting lipid-rich plaque has been verified in bench tests using a plaque model made by injecting lard into excised swine carotid arterial vessel. The plaque models are imaged in water at working distances of 0 to 2 mm, and significantly distinguished from normal vessels.

Fluorescence labeled biomarkers can be detected during endoscopy to guide early cancer biopsies, such as high-grade dysplasia in Barrett's Esophagus. To enhance intraoperative visualization of the fluorescence hot-spots, a mosaicking technique was developed to create full anatomical maps of the lower esophagus and associated fluorescent hot-spots. The resultant mosaic map contains overlaid reflectance and fluorescence images. It can be used to assist biopsy and document findings. The mosaicking algorithm uses reflectance images to calculate image registration between successive frames, and apply this registration to simultaneously acquired fluorescence images. During this mosaicking process, the fluorescence signal is enhanced through multi-frame averaging. Preliminary results showed that the technique promises to enhance the detectability of the hot-spots due to enhanced fluorescence signal.

Here, we present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance modality and high-spatial resolution (~ 0.5 um) multiphoton imaging. The essential element of the endoscope is a 3 mm outside diameter (OD), catadioptric zoom lens based on the idea of separating the optical paths of excitation light with different wavelengths. The two imaging modes are switched by changing the wavelength of the excitation light and, therefore, the optical zoom operation is achieved without any mechanical adjustment at the endoscope distal end. We aligned in free space the zoom lens with a previously demonstrated miniaturized resonant/non-resonant fiber raster scanner. We tested the performance and confirmed the high resolution and large FOV of this miniature device by imaging a US Air Force test target in transmission. We acquired ex vivo images of unstained rodent tissues by using the large FOV mode to navigate to the site of interest and then using the high resolution modality to image with cellular details. The demonstrated endomicroscope with optical zoom capability is a significant step toward developing clinical optical tools for real time tissue diagnostics.

This work deals with label free multiphoton imaging of the human lung tissue extra-cellular matrix (ECM) through optical fibers. Two devices were developed, the first one using distal scanning associated to a double clad large mode area (LMA) air-silica microstructured fiber, the second one using proximal scanning of a miniature multicore image guide (30000 cores inside a 0.8 mm diameter). In both cases, the main issue has been efficient linear and nonlinear distortion pre-compensation of excitation pulses. By inserting before the delivery fiber a compact (10 cm × 10 cm footprint) grisms-based stretcher (a grating in close contact with a prism) made of readily available commercial components, we achieved as short as 35-femtosecond-duration pulses that were temporally compressed at the direct exit of a 2-meter-long fiber. Interestingly, this femtosecond pulse fiber delivery device is also wavelength tunable over more than 100 nm inside the Ti: Sapphire emission band. With the help of distal scan system, those unique features allowed us to record elastin (through two-photon fluorescence) and collagen (through second harmonic generation) fibered network images. These images were obtained ex-vivo with only 15 mW @ 80 MHz of IR radiation delivered to the alveoli or bronchus tissues. 3D imaging with 400-μm-penetration depth inside the tissue was possible working with a 2-meter-long LMA fiber. With the help of proximal scanning, the miniature image guide allowed us to perform endoscopic real time microimaging of the ECM ex vivo.

We have previously developed side-viewing endoscopic OCT systems to detect colorectal cancer in the murine model, which longitudinally scans the mouse colon at 8-16 discrete angular positions. This small number of angles is chosen to keep imaging time and the amount of data to analyze reasonable, but this azimuthal undersampling of the tissue may result in missed or incorrectly characterized adenomas. A need exists for a spiral-scanning OCT endoscope capable of generating 3D, in vivo OCT data sets that satisfy the Nyquist criterion for adequate sampling of the tissue. Our new endoscopic system replaces the sample arm optics of a commercial OCT system with a spiral-scanning, gradient-index lens-based endoscope. The endoscope provides unit magnification at a working distance capable of producing a focal depth of 280 μm in tissue. The working distance accounts for a 41° rod prism that reflects the beam through the endoscopic window into the tissue while minimizing back reflection. A swept-source laser with a central wavelength of 1040 nm and spectral bandwidth of 80 nm provides an axial resolution of 12 μm in air and 9 μm in water. The endoscope has a theoretical diffraction-limited lateral resolution of 5.85 μm. We present fully sampled, 3D, in vivo images of the mouse colon.

The accepted model of colorectal cancer assumes the paradigm that aberrant crypt foci (ACF) are the earliest events in tumorigenesis and develop into adenoma, which further develop into adenocarcinoma. Under this assumption, basic research and drug studies have been performed using ACF as substitute markers for fully developed carcinoma. While studies have shown a correlation between the number of ACF present and the presence of adenoma/adenocarcinoma, a causal relationship has yet to be determined. The mouse has shown to be an excellent model for colorectal cancer; however, the outcomes of such experiments require sacrifice and histologic examination of ex vivo tissue. To better utilize the mouse model to study ACF and adenoma development, an endoscope was constructed for non-destructive in vivo surface visualization, molecular imaging and cross-sectional imaging of the colon. Our system combines surface magnifying chromoendoscopy (SMC) and optical coherence tomography (OCT) to image colon microstructure. Sixteen mice, treated with the carcinogen azoxymethane, were imaged at 2 week intervals, to visualize carcinogenesis events. With this dual-modality system we are able to visualize crypt structure alteration over time as well as adenoma development over time.

Our work demonstrates a MEMS based handheld dual-axis confocal microscope for cervical cancer screening. Imaging demonstration is performed with plant and animal tissue biopsies. The data is collected and displayed in real time with 2-5 Hz frame rates.

Minimally invasive surgical techniques for endoscope become widely used, for example, laparoscopic operation, NOTES (Natural Orifice Translumenal Endoscopic Surgery), robotic surgery and so on. There are so many demand and needs for endoscopic diagnosis. Especially, palpation is most important diagnosis on any surgery. However, conventional endoscopic system has no tactile sensibility. There are many studies about tactile sensor for medical application. These sensors can measure object at a point. It is necessary to sense in areas for palpation. To overcome this problem, we propose compound eye type tactile endoscope. The proposed system consists of TOMBO (Thin Observation Module by Bound Optics) and clear silicon rubber. Our proposed system can estimate hardness of target object by measuring deformation of a projected pattern on the silicon rubber. The purpose of this study is to evaluate the proposed system. At first, we introduce approximated models of the silicone and the object. We formulate the stiffness of object, the deformation of silicone, and the whole object. We investigate the accuracy of measured silicone’s lower surface for deformation of silicone by prototype system. Finally, we evaluate the calculated stiffness of the soft object.