In the present study, we investigated the feasibility of employing multiphoton microscopy as a noncontact in situ imaging tool for determination of RF diffusion profile and evaluation of CXL efficacy. We further provided evidence of morphofunctional changes of cross-linked corneas as key references to ascertain the sensitivity and fidelity of SHG imaging for detecting CXL-induced alterations. Since corneal collagen organization exhibits significant interspecies differences,
17 we used human corneas in order to obtain the most clinically relevant data. Despite the fact that CXL was proposed as a promising procedure for treating ectatic corneal disorders in the early and intermediate disease stages more than a decade ago,
1,2 a clinical tool providing reliable assessment of early CXL therapeutic efficacy and capable of predicting long-term treatment outcomes is unavailable so far. Current clinical parameters to determine CXL efficacy mainly rely on measuring corneal optical properties such as refractive power by autorefractor and wavefront aberrometry, uncorrected and best-corrected visual acuity, as well as corneal physical properties such as topography by photokeratoscopy and tomography by anterior segment optical coherence tomography (AS-OCT).
17,23,24 Nevertheless, none of these conventional methods is sensitive enough to reveal subtle changes of lamellar collagen organization caused by CXL at an ultrastructural level, neither immediately nor during long-term follow-ups after RF-UVA–based phototherapy. A new technique combining corneal pachymetry and topography, known as Scheimpflug imaging, was recently introduced to evaluate the clinical efficacy of CXL.
25 Although this imaging technique offers information on both refractive and physical changes in cross-linked corneas, depth-dependent lamellar structural changes of the cornea are not resolved and the functional significance of such measurements remains to be validated by long-term clinical outcomes. Although conventional OCT has been extended to study anterior corneal structures (i.e., AS-OCT), this method is not sensitive enough to reveal collagen architectural alterations following CXL. In contrast to these currently applied clinical measurements, the noninvasive in situ multiphoton microscopy, offering insightful structural information at a supramolecular level, has recently been used to study CXL effects in several animal models.
12–16,26 Second harmonic generation, a key imaging module of multiphoton microscopy, has been shown to depict detailed fibrillar collagen ultrastructure of corneas.
14 In agreement with previously reported data from Gupta et al.
16 in porcine eyes, we observed an increased homogeneity in stromal collagen texture and a reduced roughness indexed by declined Rq values of cross-linked human corneal stroma (
Fig. 3). Decreased Rq values of SHG images reflect the en face morphological changes from densely packed, parallel collagen bundles to interwoven, homogenous, and short bands of collagen fibrils, indicating an increased number of collagen cross-links in RF-UVA–treated stroma. Interestingly, the roughness profiles of cross-linked corneas showed the maximal reduction of Rq values mainly in the middle segment of corneal stroma in all RF doses studied (
Fig. 3,
Supplementary Table S3). Our findings are in line with quantitative studies employing different imaging modalities. Using two-photon collagen autofluorescence microscopy, Chai et al.
12 stated that CXL-induced alterations of collagen autofluorescence intensities were confined to the anterior 220 to 280 μm of lapine corneal stroma, independent of administered RF doses. By Brillouin optical microscopy, Scarcelli et al.
13 detected an anterior segment–defined Brillouin frequency shift in cross-linked bovine corneas. Compared with the aforementioned imaging techniques, SHG microscopy has the advantage of superior resolution by delineating the lamellar collagen ultrastructure as a noninvasive therapeutic parameter. Despite the compelling difference in average tissue roughness between native and RF-UVA cross-linked corneas, we did not detect any differences within treatment groups (
Fig. 3C). Taken together, these data indicate a high sensitivity of SHG multiphoton microscopy to CXL-induced alterations in human corneas, while the specificity of SHG in determining the magnitude and dimension of structural modifications caused by different doses of RF remains suboptimal. A plausible explanation for this observation is that the laser intensity of multiphoton microscope is intrinsically attenuated in deeper stroma,
27 thereby impeding the power of SHG microscopy to discriminate subtle CXL changes within treatment groups.