Depth-resolved fluorescence lifetime spectroscopy across the cornea in the digital frequency domain

The cornea is the primary refractive component of the eye with a central connective tissue (stroma) which is bounded by stratified epithelium (forms the anterior surface) and endothelium (forms the posterior monolayer). It is transparent to visible light and hence amenable to fluorescence spectroscopy. Here we report on developing a dedicated ophthalmic time-resolved confocal scanning microfluorometer (OTR-CSMF) for depth-resolved transcorneal spectroscopy. The instrument combines a confocal microfluorometer, nanostage, digital frequency domain unit for rapid lifetime acquisition, and a corneal perfusion system. Using a 40x objective (0.8 NA; wd = 3.3 mm; water), the instrument offers depth scanning with an axial resolution of similar to 1.3 mu m and single-molecule detection sensitivity, validated by correlation spectroscopy with fluorescein (100 pM). In addition, the device can resolve fluorescence lifetimes from 100 ps to 100 ms. With porcine corneas ex vivo, topical administration of Rhodamine B showed fluorescence peaks in the epithelium and endothelium consistent with its lipophilicity and consequent accumulation in the cellular layers. Moreover, the high axial resolution of the instrument revealed (a) fluorescence discontinuities at the interface between epithelium and stroma and between stroma and endothelium, and (b) fluorescence spikes in the stroma corresponding to dye accumulation in the keratocytes. These data indicate transcorneal transport of Rhodamine B by sequential diffusion and partitioning. The lifetime of Rhodamine B revealed characteristic variations across the depth, potentially due to variable dye accumulation and/or local variations in the refractive index. Similar experiments with the relatively hydrophilic fluorescein showed high fluorescence in the stroma compared to the cellular layers. Overall, OTR-CSMF is optimized for transcorneal fluorescence spectroscopy. Accordingly, we envision applying the device to register transcorneal dynamics of pO(2), temperature, pH, fluorescent drug surrogates, and electrolytes, among other parameters in the future. These measurements can be expected to revitalize investigations related to drug discovery and the pathophysiology of corneal disorders.