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Dagny Zhu, Cosimo Arnesano, Jiun Lap Do, Grace Shih, Jessica Cao, Scott Fraser, Martin Heur; Functional Imaging of Ex Vivo Human Corneal Endothelium Using Two-Photon Fluorescence Lifetime Imaging Microscopy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1937.
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© ARVO (1962-2015); The Authors (2016-present)
The global state of corneal endothelial health is currently assessed with endothelial cell density and morphology using specular microscopy. Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) is a powerful non-contact imaging modality that can be used to characterize the metabolic activity of living tissue by measuring the auto-fluorescence of endogenous biomolecules essential for cellular function. Herein, we characterized the metabolic state of ex-vivo human corneal endothelial cells by imaging free and protein-bound nicotinamide adenine dinucleotide (phosphate) (NAD(P)) under different stress conditions.
Human donor corneas not suitable for transplantation were purchased from VisionShare and treated with vehicle control (n=2) or acetazolamide (n=2). A band-pass filter of 460/80nm was used for NAD(P) detection. Fluorescence decay time (lifetimes) of protein-bound and free NAD(P) in endothelial cells were imaged and analyzed at t = 0, 5, 15, and 30 min. Fluorescence lifetime images were acquired with a Zeiss LSM-780 inverted confocal microscope coupled to a Ti:Sapphire laser system (Coherent Chameleon Ultra II) and an ISS A320 FastFLIM. FLIM data were acquired by the VistaVision software from ISS and processed by SimFCS software.
2P-FLIM images of ex-vivo human corneal endothelium showed that the ratio of protein-bound to free NAD(P) in corneas treated with vehicle control remained unchanged over time. Treatment with acetazolamide, a carbonic anhydrase inhibitor known to play a role in corneal endothelial dysfunction, resulted in a shift from free to protein-bound NAD(P) over time, reflecting an overall reduction in cellular metabolism and ATP production that is consistent with previous results obtained with other tissues under stress conditions.
FLIM’s potential as a clinical optical imaging modality has already been described for diseases of the human retina and corneal epithelium. This study demonstrates proof of principle of using 2P-FLIM for non-invasive functional imaging of live human corneal endothelium. Future refinements could lead to further research and clinical applications for evaluation of corneal endothelial function under various pathophysiological conditions.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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