For in-vivo imaging applications the use of conventional confocal microscope systems are simply not practical due to their relatively large size and weight. There is, however, great interest from both the life science research community and the clinical profession for the development of compact and portable micro-optical instrumentation capable of achieving minimally invasive, in-vivo imaging of tissue with sub-cellular resolution. In this paper we describe a novel confocal micro-imaging system incorporating, at its core, a thermally driven, non-resonant two-axis MEMS scanner which serves as a substitute for the two single-axis galvanometer scanners commonly used in standard confocal imaging systems. In this paper we describe the non-linearity of such devices and a number of techniques to compensate for this.

We designed and constructed a two-fiber-optic two-photon endoscope with compact two axes piezo scanner system and a miniature high 0.65 NA Grin lens objective. The high NA GRIN lens objective coupled to the fibers with a beam splitter cube represent a rigid miniaturized optical system block which is scanned as a whole by a piezo scanner allowing always an on-axes beam irradiation of the optical system. We have demonstrated the ability of our system to perform good images in the micrometer field. Fluorescence detection was able at different wavelengths between 790 and 840 nm which could allow SHG detection. The endoscope is high flexible and controllable in terms of time acquisition, resolution and magnification. Fluorescence images were acquired over a maximal 420 μm x 420 μm field-of-view.

Spectrally encoded confocal microscopy (SECM) and optical frequency domain imaging (OFDI) are two reflectancebased imaging technologies that may be utilized for high-resolution microscopic screening of internal organs. SECM provides en face images of tissues with a high lateral resolution of 1-2 μm, and a penetration depth of up to 300 μm. OFDI generates cross-sectional images of tissue architecture with a resolution of 10-20 μm and a penetration depth of 1- 2 mm. Since the two technologies yield complementary microscopic information on two different size scales (SECM-cellular and OFDI-architectural) that are commonly used for histopathologic evaluation, their combination may allow for more accurate optical diagnosis. Here, we report the integration of these two imaging modalities in a single bench top system. SECM images of swine small intestine showed the presence of goblet cells, and OFDI images revealed the finger-shaped villous architecture. In clinical study of 9 gastroesophageal biopsies from 8 patients, a diverse set of architectural and cellular features was observed, including squamous mucosa with mild hyperplasia and gastric antral mucosa with gastric pits and crypts. The capability of this multimodality device to enable the visualization of microscopic features on these two size scales supports our hypothesis that improved diagnostic accuracy may be obtained by merging these two technologies into a single instrument.

Optical Coherence Microscopy (OCM) combines coherence gating, high numerical aperture optics, and a fiber core pinhole to provide high axial and lateral resolution with relatively large depth of imaging. We present a handheld rigid OCM endoscope with a 6 mm diameter tip, 1 mm scan width, and 1 mm imaging depth. This probe will allow noninvasive imaging of fine structural detail in vivo. X-Y scanning is performed distally with mirrors mounted to micro galvonometer scanners incorporated into the endoscope handle. Two scanning doublet lenses relay the stop from the galvonometers to the afocal relay stop. The endoscope optical design consists of an afocal Hopkins relay lens system and a 0.4 NA objective. To allow focusing at various depths in the tissue, the endoscope housing is designed in two pieces screwed together with a fine pitch threads. A small rotation of the outer housing moves the lenses proximal and distal relative to the window, causing the focal location in the tissue to change. The space between the final objective lens and the window is filled with distilled water to avoid misalignment of the focus and coherence gate. A knife edge test was performed and the line spread function FWHM was measured to be 2.25 μm. The MTF has at least 0.3 contrast at a 5 μm line pair. This rigid handheld OCM endoscope will be useful for application ranging from minimally invasive surgical imaging to assessing dysplasia and sun damage in skin.

Background: Confocal Laser Endomicroscopy (CLE) based on ultraminiature miniprobes (Cellvizio®, Mauna Kea Technologies, Paris, France) is able to image the inner microstructure of retroperitoneal full organs punctured during EUS-FNA procedures, such as pancreas, liver or lymph nodes. Therefore, pCLE can provide an easy-to-use and precise adjunct tool to ultrasonographic interventions in order to target suspicious areas for biopsies in EUS-FNA. Material and Methods: Probe-based CLE (pCLE) was performed on ex-vivo surgically resected specimens after topical application of fluorophores in standard 19G and 22G needles. Two prototype miniprobes ("S-probe" 300 microns diameter, field of view 400*280 microns, and "S-probe" 650 microns diameter, field of view 500*600 microns) were then inserted into the needles and enabled visualization of the inner microstructures of uterus, lung, kidney, stomach and esophagus, in both healthy and cancerous conditions. Then, pCLE was performed in-vivo on four pigs during three NOTES and one EUS-FNA procedures after intravenous injection of 2-7mL fluorescein 1-10% using the prototype "S-probe" 350 microns diameter inserted in 19G FNA needles. Liver, pancreas and spleen were imaged. Results: During the ex-vivo experiments, pCLE made it possible to distinguish microstructures, such as alveoli and macrophages in the lungs. During the in-vivo experiments, Cellvizio® video sequences showed hepatic lobules and the portal vein in the liver, and red and white pulp in the spleen. Conclusion: pCLE provides in vivo cellular information about full organs. It has the potential to help target biopsies during EUSFNA, which suffers from a high rate of false negatives, thus increasing its sensitivity.

We previously reported on the development of a multi-spectral confocal laparoscope for clinical imaging. In this paper we present current results using the system to image ovaries with a new laparoscope design using the contrast agent acridine orange. This new laparoscope integrates computer controlled systems for focus, depth scans, and localized contrast agent delivery. Precise axial position control is accomplished with tiny stepper motors integrated inside the laparoscope handle. Ergonomic handle controls allow for data acquisition, deliver of contrast agents, and adjustment of imaging depth during procedures by the surgeon. We have approval to use acridine orange in our clinical trials to image ovaries in vivo during oophorectomies. We present in vivo results using both acridine orange and fluorescein as the topically administered contrast agent.

Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

We demonstrate the proof of concept for use of a fiber optic FTIR instrument to perform in vivo detection of colonic neoplasia as an adjunct to medical endoscopy. FTIR is sensitive to the molecular composition of tissue, and can be used as a guide for biopsy by identifying pre-malignant tissue (dysplasia). First, we demonstrate the use of a silver halide optical fiber to collect mid-infrared absorption spectra in the 950 to 1800 cm^-1 regime with high signal-to-noise from biopsy specimens of colonic mucosa tissue ex vivo. We observed subtle differences in wavenumber and magnitude of the absorbance peaks over this regime. We then show that optimal sub-ranges can be defined within this spectral regime and that spectral pre-processing can be performed to classify the tissue as normal, hyperplasia, or dysplasia with high levels of performance. We used a partial least squares discriminant analysis and a leave-one-subject-out crossvalidation strategy to classify the spectra. The results were compared with histology, and the optimal thresholds resulted in an overall sensitivity, specificity, accuracy, and positive predictive value of 96%, 92%, 93%, and 82%, respectively for this technique. We demonstrate that mid-infrared absorption spectra can be collected remotely with an optical fiber and used to identify colonic dysplasia with high accuracy. We are now developing an endoscope compatible optical fiber to use this technique clinically for the early detection of cancer.

We present a kind of rotational two photon mciroendoscopy for 1μm fiber femtosecond laser. The fiber laser provide ultrashort femto-second pulses with center wavelength at 1.034μm and repetition rate of 50MH. The rotational probe is based on double cladding photonic crystal fiber (CD PCF) fiber, Grin lens, microprism and rotational MEMS motor. The MEMS motor has diameter of 2.2mm and can provide 360 degree full view rotation. We experimentally show that the DC PCF fiber works for 1μm fiber laser two photon system. Second harmonic generation (SHG) singnal line profile of rat tail tendon and fish scale was taken with the endoscopy system.