Assessment of the corneal collagen organization after chemical burn using second harmonic generation microscopy

Abstract

The organization of the corneal stoma is modified due to different factors, including pathology, surgery or external damage. Here the changes in the organization of the corneal collagen fibers during natural healing after chemical burn are investigated using second harmonic generation (SHG) imaging. Moreover, the structure tensor (ST) was used as an objective tool for morphological analyses at different time points after burn (up to 6 months). Unlike control corneas that showed a regular distribution, the collagen pattern at 1 month of burn presented a non-organized arrangement. SHG signal levels noticeably decreased and individual fibers were hardly visible. Over time, the healing process led to a progressive re-organization of the fibers that could be quantified through the ST. At 6 months, the stroma distribution reached values similar to those of control eyes and a dominant direction of the fibers re-appeared. The present results show that SHG microscopy imaging combined with the ST method is able to objectively monitor the temporal regeneration of the corneal organization after chemical burn. Future implementations of this approach into clinically adapted devices would help to diagnose and quantify corneal changes, not only due to chemical damages, but also as a result of disease or surgical procedures.

Structural changes in the crystalline lens as a function of the postmortem interval assessed with two-photon imaging microscopy

“…Multiphoton microscopy has provided very useful information when used to image ocular tissues. This technique has been able to detect temporal variations in corneas with swelling and edema [ 33 , 34 ], after chemical burn [ 35 ] and in post-crosslinking conditions [ 36 , 37 ]. Moreover, lifetime autofluorescence has also been evaluated in human corneas before transplantation [ 38 ].…”

Two-Photon Imaging for Non-Invasive Corneal Examination

“…Several approaches have been proposed to retrieve this information and distinguish between tissues with different levels of organization. These include gray level co-occurrence matrix (GLCM) [92,93,100,101], structure tensor (ST) [102][103][104][105], and Fourier Transform (FT) analysis [101,[106][107][108][109][110][111][112][113][114]. FT-based analysis is a popular and well-established approach.…”

“…Metabolic changes during corneal wound healing, based on NAD(P)H AF lifetime, were also monitored by Gehlsen and coworkers [179]. Recently, SHG imaging combined with ST for quantitative evaluation of collagen organization was employed to monitor corneal stroma architecture following chemically induced burns [103]. According to the authors, normal levels of collagen organization can be reached 6 months after burning [103].…”

On the quantitative analysis of lamellar collagen arrangement with second-harmonic generation imaging

“…FT analysis has been used to confirm the expected differences in organization levels between healthy, keratoconus, and crosslinked corneas [ 9 , 11 ], to compare the organization levels of corneal samples with different storage times [ 37 ], to assess changes in injured tendons [ 42 ], or to characterize breast [ 43 ] and ovarian [ 44 , 45 ] tumorous tissue. First introduced by Ávila et al in 2015 to assess the organization of collagenous tissue [ 38 ], ST has already demonstrated to have the potential to differentiate between healthy and diseased corneas [ 40 ], crosslinked and non-crosslinked corneas [ 12 ] and to monitor corneal healing after chemical burns [ 39 ]. GLCM has been shown to be a viable tool for monitoring the decrease in collagen organization expected during corneal storage [ 37 ], in the evaluation of ovarian tumors [ 5 , 44 ], and in the characterization of healthy dermis and scar tissue [ 2 ].…”