651 Targeted Vertical Cross-Sectional Imaging With Near-Infrared Dual Axes Confocal Fluorescence Endomicroscope

Abstract

Background: Pathologists review histology cut perpendicular to the tissue surface or in the vertical cross-section in order to visualize the normal or abnormal differentiation pattern of tissue. Unlike other endomicrosope designs, the dual axes confocal architecture has sufficient dynamic range to image in this orientation. Dysplasia is a pre-malignant condition in the colon that can progress into colorectal cancer. Peptides have demonstrated tremendous potential for in vivo use to detect colonic dysplasia and can be labeled with near-infrared (NIR) dyes for visualizing the full depth of the epithelium. Aims: We aim to demonstrate vertical cross-sectional images over a large field-of-view (FOV) with a dual axes confocal endomicroscope of NIR fluorescence images that visualize specific peptide binding to dysplasia in the mouse colon. Methods: We use a peptide selected with phage display technology. The amino acid sequence LTTHYKL is connected on the C-terminus by a GGGSK linker to the Cy5.5 fluorophore to prevent steric hindrance, hereafter LTT*-Cy5.5. We use CPC;Apc mice that are genetically engineered to spontaneously develop pre-malignant colonic lesions that resemble that of human disease on histology. CPC;Apc mice of age 6-7 months were injected with the Cy5.5-labeled peptide, LTT*-Cy5.5, at a concentration of 400 μM via tail vein. The peptide conjugate was allowed to circulate for 15 minutes. The mice were then euthanized, and the colon was excised, washed, blotted dry, and mounted on glass slides using 3M Vetbond tissue adhesive. The fresh colonic bulk tissues were imaged immediately with 2 mW of excitation at wavelength 671 nm. The tissues were then fixed with 10% buffered formalin for 24 hours, paraffin-embedded and sectioned into 10 μm thin slices and stained with hematoxylin and eosin (H&E). Results: NIR fluorescence images of colonic mucosa show dysplastic crypts in the vertical orientation, demonstrating the histology-like performance over a FOV of 800 μm (width)×400 μm (depth). The contrast from specific binding of the LTT-Cy5.5 peptide can be appreciated by the image of the border between normal colonic mucosa and dysplasia, Fig. a. Vertically oriented crypts can be appreciated in the corresponding histology (H&E), scale bar 200 μm, Fig. b. Use of a specific binding peptide allows for the transition from normal to pre-malignant mucosa to be visualized with high contrast at the same Photomultiplier tube (PMT) gain setting. Compared to normal colonic mucosa, the NIR fluorescence intensity of dysplasia is greater by at least a factor of 3. Conclusions: We demonstrate vertical cross-sectional imaging using a novel dual axes confocal endomicroscope based to collect NIR fluorescence images over a 800 μm ×400 μm field-of-view to detect Cy5.5-labeled peptides that bind specifically to pre-malignant colonic mucosa.