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Jared L. Matthews, Shradha Prabhulkar, Adam de la Zerda, Sanjiv Gambhir, Richard Awdeh; Molecular Histopathology Using Gold Nanorods And Optical Coherence Tomography. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5617.
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© ARVO (1962-2015); The Authors (2016-present)
This study describes the novel application of commercially available OCT systems towards molecular histopathology of conjunctival cancers (squamous cell carcinoma). We have demonstrated that optimized gold nanorods (GNRs) produce a strong OCT signal, beyond the background tissue OCT signal, this fulfilling the role of a stain or reporter moiety. The advantage of using GNRs is that it does not require further development, such as the commonly used enzyme stains, and is more robust, stable, and resistant to photo-bleaching, as compared to fluorophores. The GNRs are linked to anti-Glut-1 antibodies which act as the biorecognition molecules. Glut-1 is chosen as the model analyte as it has been shown to be overexpressed at the surface of certain ocular surface tumors.
GNRs with a longitudinal plasmon resonance at 840nm were synthesized using seed-mediated growth mechanism and covalently attached to anti-GLUT-1 antibodies via carbodiimide chemistry. 10µm sections of paraffin-embedded conjunctival tissue from human subjects were de-paraffinized, re-hydrated, blocked and antigen retrieval was performed as per standard pathology laboratory protocol. The tissue samples were then incubated with 10nM of anti-Glut-1 tagged gold nanorods for a period of 45 minutes and trice rinsed. OCT imaging was then performed using a rastor scanning protocol covering an area of 4 X 4mm. Every imaging session consisted of 100 B-scans (800 A-scans/B-scan). En-face slices of OCT images were reconstructed from the 3-D datasets by averaging over 10µm in the axial direction using Matlab. Traditional immunohistochemistry staining of tissue samples for Glut-1 was performed using immunoperoxidase.
OCT images showed increased scattering in areas of tissue overexpressing Glut-1 protein as observed by the traditional immunoperoxidase staining. The intensity of scattering correlated well with the tumor grade (mild, moderate, in-situ) as per statistically analysis of six distinct patient tissue samples within each tumor grade.
We have developed novel histopathology strategy for the analysis of ocular tumors using commercially available OCT systems. Our future efforts will focus on software development to enhance image quality, automation and high throughput analysis.
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