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Aim 1: Develop single axis side-view confocal microendoscopes for pre-clinical and clinical imaging
Aim 2: Develop a multiphoton microendoscope for imaging in horizontal and vertical planes
Aim 3: Develop a front and side-view dual axes confocal microendoscope for real time in vivo imaging
Single axis confocal
A single mode optical fiber (SMF) and objective lens are aligned along the main axis of the microendoscope. In vivo fluorescence images are collected with sub-cellular resolution. In the side-view geometry, the focusing optics can be placed in direct contact with the mucosal surface in hollow organs. The beam exits a SMF, and is collimated and focused by lenses. A monolithic 3-axis scanner/actuator developed in TR&D1 will be located in the post-objective position. A solid immersions lens will couple the beam into the tissue. The optics and scan mirror will be packaged into an instrument with a with a side-view configuration. The scan mirror can collect images in either the horizontal or vertical plane, and 3D volumetric images can be generated by combining scanning with a pullback of the instrument. This configuration provides the smallest diameter instrument for use in areas that are difficult to access, such as oral cavity.Single axis confocal
Multi-photon
Ultra-fast pulses delivered via a photonic bandgap fiber are deflected by a polarization beam splitter (PBS) through a quarter-wave plate (QWP), and focused by an aspheric lens onto an actuator. The beam reflects off the reflective surface with a 180o phase shift, and is displaced in the axial direction. The polarized beam passes back through QWP, and is relayed using achromatic doublets onto the lateral scanner. Fluorescence is collected, deflected by the dichroic mirror (DM), and is focused by an achromatic doublet into a multi-mode fiber for detection by a PMT. Axial displacement is achieved using an electrothermal mirror, developed in TR&D1. The reflector is supported by 4 legs that have inner and outer folded U-shaped actuators. Each leg has 4 embedded heaters connected in series made-up of thin platinum (Pt) wires. The bimorph layers formed by silicon oxide and aluminum bends on heating due to a difference in thermal expansion coefficient.Multi-photon
Dual axes confocal
Two separate collimators direct light through a low NA objectives, and are oriented at an angle to the main optical axis. The region where the illumination and collection paths overlap defines the focal volume, and significantly reduces the resolution. Light scattered by tissue along the illumination path enters the collection optics at large angles and substantially reduces the likelihood of capture. This effect improves the dynamic range so that images can be collected in vertical cross-sections as well as horizontal. The low NA objectives create a long working distance so that the scanning mechanism can be placed in the post-objective position. This configuration allows for scalability of the optical design so that the instrument diameter be substantially reduced without loss of resolution. Also, images to be collected with a much larger FOV. A solid immersion lens (SIL) is located on the sidewall to couple light into the tissue.Dual axes confocal
Design, configuration, and performance of TR&D2 microendoscopes. Parameters are shown for the expected imaging performance of flexible fiber-coupled microendoscopes that have been developed by TR&D2.
Single axis confocal. A 4.2 mm diameter instrument was inserted into mouse colon on a colonic adenoma in vivo. NIR fluorescence image was collected in the vertical (XZ) plane with a FOV of 600×292 um2. A Cy5.5-labeled peptide specific for EGFR (300 mM, 200 mL of PBS) was administered intravenously ~1.5 hour beforehand. Epithelial structures and cells can be visualized, including crypt (arrow), lumen (l), goblet cells (g), cytoplasm (c), immune cells (arrowhead), lamina propria (lp).
Multi-photon. A microsystems actuator was used to collect a fluorescence image ex vivo in the vertical (XZ) plane with an axial depth >200 um. The horizontal width is 270 um. The colon specimen was resected from a mouse genetically engineered to constitutively express tdTomato. Numerous epithelial structures and individual cells can be visualized in this orientation, including crypt (arrow), lumen (l), goblet cells (g), cytoplasm (c), immune cells (arrowhead), lamina propria (lp).
Dual axes confocal. A 5.5 mm diameter front view instrument was placed on the epithelial surface of a colonic adenoma ex vivo. NIR fluorescence image was collected in the vertical (XZ) plane with a FOV of 1000×430 um2. The specimen was resected from a mouse genetically engineered to delete the APC gene and spontaneously form adenomas. A Cy5.5-labeled peptide specific for EGFR (300 mM, 200 mL of PBS) was administered intravenously ~1.5 hour beforehand.
Publications
Lee M, Li G, Li H, Duan X, Birla MB, Chang TS, Turgeon DK, Oldham KR, Wang TD. Confocal laser endomicroscope with distal MEMS scanner for real-time histopathology. Scientific Reports 2022;12:1-11. PMC9684518.
Lee M, Li H, Birla MB, Li G, Wang TD, Oldham KR. Capacitive Sensing for 2-D Electrostatic MEMS Scanner in a Clinical Endomicroscope. IEEE Sensors Journal 2022;22:24493-503. IEEE
Li G, Duan X, Lee M, Birla M, Chen J, Oldham KR, Wang TD, Li H. Ultra-Compact Microsystems-Based Confocal Endomicroscope. IEEE Transactions on Medical Imaging 2020;39:2406-14. PMC7918297.
Li G, Li H, Duan X, Zhou Q, Zhou J, Oldham KR, Wang TD. Visualizing Epithelial Expression in Vertical and Horizontal Planes With Dual Axes Confocal Endomicroscope Using Compact Distal Scanner. IEEE Transactions on Medical Imaging 2017;36:1482-90. PMID: 28252391.
Gao Z, Li G, Li X, Zhou J, Duan X, Chen J, Joshi BP, Kuick R, Khoury B, Thomas DG, Fields T, Sabel MS, Appelman HD, Zhou Q, Li H, Kozloff K, Wang TD. In vivo near-infrared imaging of ErbB2 expressing breast tumors with dual-axes confocal endomicroscopy using a targeted peptide. Scientific Reports 2017;7:14404. PMC5663926.
Duan X, Li H, Li X, Oldham KR, Wang TD. Axial beam scanning in multiphoton microscopy with MEMS-based actuator. Opt Express 2017;25:2195-2205. PMC5772401.
Duan X, Li H, Wang F, Li X, Oldham KR, Wang TD. Three-dimensional side-view endomicroscope for tracking individual cells in vivo. Biomedical Optics Express 2017;8:5533-5545. PMC5745101.
Duan X, Li H, Zhou J, Zhou Q, Oldham KR, Wang TD. Visualizing epithelial expression of EGFR in vivo with distal scanning side-viewing confocal endomicroscope. Scientific Reports 2016;6:37315. PMC5118792.
Duan X, Li H, Qiu Z, Joshi BP, Pant A, Smith A, Kurabayashi K, Oldham K, Wang TD. MEMS-based multiphoton endomicroscope for repetitive imaging of mouse colon. Biomedical Optics Express 2015;6:3074-82. PMC4541532.
Qiu Z, Khondee S, Duan X, Li H, Mandella MJ, Joshi BP, Zhou Q, Owens SR, Kurabayashi K, Oldham K, Wang TD. Vertical cross-sectional imaging of colonic dysplasia in vivo with multi-spectral dual axes confocal endomicroscopy. Gastroenterology 2014;146:615-17. PMC4029422.
Qiu Z, Liu Z, Duan X, Khondee S, Joshi BP, Mandella MJ, Oldham K, Kurabayashi K, Wang TD. Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope. Biomedical Optics Express 2013;4:322-330. PMC3567718.
Piyawattanametha W, Ra H, Qiu Z, Friedland S, Liu JTC, Loewke K, Kino GS, Solgaard O, Wang TD, Mandella MJ, Contag CH. In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract, J Biomedical Optics 2012:17:021102. PMC3380818.
Elahi SF, Liu Z, Luker KE, Kwon RS, Luker GD, Wang TD. Longitudinal Molecular Imaging with Single Cell Resolution of Disseminated Ovarian Cancer in Mice with a LED-based Confocal Microendoscope. PMC3502045.
Elahi SF, Miller SJ, Joshi B, Wang TD. Targeted imaging of colorectal dysplasia in living mice with fluorescence microendoscopy. Biomedical Optics Express 2011;2:981-6. PMC3072136.
Piyawattanametha W, Wang TD. MEMS-Based Dual-Axes Confocal Microendoscopy. IEEE J Sel Topics Quantum Electronics 2010;16:804-14. PMC3242380.
Piyawattanametha W, Ra H, Mandella MJ, Loewke K, Wang TD, Kino GS, Solgaard O, Contag CH. 3-D Near Infrared Fluorescence Imaging using a MEMS-based miniature Dual-Axis Confocal Microscope. IEEE J Sel Topics Quantum Electronics 2009;15:1344-50. IEEE.
Wong LK, Mandella MJ, Kino GS, Wang TD. Improved rejection of multiply scattered photons in confocal microscopy using dual-axes architecture, Optics Letters 2007;32:1674-6. PMC2104523.
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