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The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband source at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4 to 5-μm resolution, at tissue depths up to 610 μm. Post-objective scanning enables a large field of view (610 x 640 μm) and balanced-heterodyne detection provides sensitivity to collect vertical sections at two frames per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect pre-cancerous tissues, and hence to perform in vivo histopathology.

A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband laser at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4- to 5-microm resolution, at tissue depths up to 610 microm. Post-objective scanning enables a large field of view (610 x 640 microm), and balanced-heterodyne detection provides sensitivity to collect vertical sections at one frame per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect precancerous tissues, and hence to perform in vivo histopathology.

We demonstrate a dual-axes confocal reflectance microscope in a 10-mm diameter handheld package. Miniaturization is achieved by using a barbell-shaped, gimbaled, two-dimensional MEMS scanner that is actuated by self-aligned vertical comb actuators. The maximum DC optical scan angles are plusmn3.3deg on the inner axis and plusmn1.0deg on the outer axis, and the corresponding resonance frequencies are 3.54 kHz and 1.1 kHz. The imaging rate is 30 frames/second, and the microscope achieves full-width-half-maximum (FWHM) transverse and axial resolutions of 8 mum and 10 mum, respectively

The application of conventional confocal microscopes with high numerical aperture (NA) to in vivo imaging is limited by the objectiveis large physical dimensions and short working distance. We are developing a confocal microscope that uses simple low NA lenses oriented in a dual axes configuration for miniaturization and in vivo imaging. This architecture achieves a long working distance, micron level axial resolution, and reduced noise from scattered light outside the focal volume. Combined with the novel method of post-objective scanning, this design can be scaled down to millimeter dimensions. We derive the dual axes response from diffraction theory, and construct two tabletop prototypes to demonstrate the performance of this approach. We collect images from freshly excised biopsy specimens of human esophagus and transgenic mouse cerebellum expressing GFP. With horizontal cross-sectional images, we achieve 1 to 2 μm resolution and collect reflectance and fluorescence images. With vertical cross-sectional images, we achieve 4 to 5 μm resolution, dynamic range of 70 dB, and tissue penetration over 1 mm. An instrument miniaturized with this configuration could be used for in vivo cellular and molecular imaging.

We present a dual-axes confocal microscope that employs postobjective scanning and low-coherence heterodyne detection to collect vertical cross-sectional images from biological tissue with high axial resolution, reduced noise from scattered light, deep tissue penetration, and a large dynamic range. This architecture can be scaled down to millimeter dimensions with microelectromechanical systems technology for performance of in vivo optical biopsy.

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle theta from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 microm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo.

A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500x500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.

We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolutionor =4.4 microm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging.

We demonstrate sequential three-dimensional in vivo imaging in one mouse, monitoring siRNA silencing with a dual-axes confocal microscope. This opens up new possibilities in developing therapeutics for genetic disorders through better assessment of treatment efficiency.