High-resolution, label-free two-photon imaging of diseased human corneas

Abstract

The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cells' metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cells' morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cells' metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases.

Quantitative Analysis of the Corneal Collagen Distribution after In Vivo Cross-Linking with Second Harmonic Microscopy

“…On the contrary, there is a significant increase in collagen organization in CXL-treated corneas whose initial distribution is partially organized or nonorganized (such as the rabbit cornea). A paradigmatic example of a nonorganized collagen arrangement is a keratoconic cornea [18–22]. At this point, further experiments on the effects of CXL treatments in keratoconic human donor corneas will help to go a step further into the understanding of CXL effects at microscopic levels.…”

“…Second harmonic generation (SHG) microscopy is a powerful nonlinear imaging technique used to visualize nonstained collagen-based structures, such as the corneal stroma [16]. SHG images of this stroma have provided information on fiber organization in healthy [15–17] and pathological samples, in particular keratoconic corneas [18–22]. Since CXL effects modify the collagen structure, SHG microscopy has been used to explore changes induced by CXL treatment.…”

“…Although the FFT has been used to quantify stromal organization in healthy and pathological corneas [20, 22, 26, 29, 30], it might have some limitations for certain collagen distributions involving crosshatching, interweaving, and wavy patterns [31]. Alternative methods include the Radon transform [32], texture analysis [27], mathematical tensorial calculus [33], gray-level cooccurrence matrices [34], and the structure tensor (ST) among others [35].…”

Quantitative Discrimination of Healthy and Diseased Corneas With Second Harmonic Generation Microscopy

“…Alterations of this intrinsic organization might seriously compromise the regular optical and metabolic functions. SHG microscopy has been reported to detect collagen structural abnormalities in corneas suffering from pathologies, such as keratoconus,10–14 keratitis,15–17 edema,18 bullous keratopathy,19,20 or fibrosis 21,22…”

Can we use rapid lifetime determination for fast, fluorescence lifetime based, metabolic imaging? Precision and accuracy of double-exponential decay measurements with low total counts

“…Although an accurate quantitative assessment of cell’s metabolism through optical techniques requires more sophisticated indicators, as the Optical Metabolic Imaging (OMI) index [3–5], the free to protein-bound ratios of the metabolic co-enzymes have been used in several studies to provide valuable information. Differences in free to protein-bound ratios associated with NADH and FAD fluorescence lifetime were already demonstrated for a wide range of biological samples and proposed as a promising diagnostic method to identify neoplastic diseases [6–8], skin diseases [9] and ocular pathologies [10,11].…”