Multimodal, multiphoton microscopy and image correlation analysis for characterizing corneal thermal damage

Abstract

We used the combination of multiphoton autofluorescence (MAF), forward second-harmonic generation (FWSHG), and backward second-harmonic generation (BWSHG) imaging for the qualitative and quantitative characterization of thermal damage of ex vivo bovine cornea. We attempt to characterize the structural alterations by qualitative MAF, FWSHG, and BWSHG imaging in the temperature range of 37 to 90 degrees C. In addition to measuring the absolute changes in the three types of signals at the stromal surface, we also performed image correlation analysis between FWSHG and BWSHG and demonstrate that with increasing thermal damage, image correlation between FWSHG and BWSHG significantly increases. Our results show that while MAF and BWSHG intensities may be used as preliminary indicators of the extent of corneal thermal damage, the most sensitive measures are provided by the decay in FWSHG intensity and the convergence of FWSHG and BWSHG images.

Analysis of spatial lamellar distribution from adaptive-optics second harmonic generation corneal images

“…Moreover the alignment of lamellae is particular for different species [6] and can be modified by different factors including surgery [11], pathologies [4,15], thermal damage [12,13], edema [29], intraocular pressure changes [30] and biomechanical conditions [18]. In this sense, an optimized quantitative method as the one here reported could help to investigate differences in stromal collagen fibers under a variety of experimental conditions.…”

“…Corneal lamellae are often organized and their orientations are often analyzed in a qualitative manner. However, quantification facilitates extraction of sensitive changes in collagen fiber organization due to pathology [4,7], surgery [8–11] or damage [12,13], and, thus may help in early diagnosis and follow-up processes.…”

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

“…In particular, SHG microscopy has been demonstrated extremely powerful to image collagen rich tissues [65] such as cornea [66,67], tendon [68,69], and arteries [70]. In particular, SHG microscopy has been mainly used for selectively investigating collagen fibres orientation and their structural changes in human dermis [71][72][73][74][75], keloid [76][77][78], fibrosis [79][80][81], thermally-treated samples [82][83][84][85][86], and also in tumour microenvironments [87][88][89][90][91][92][93]. In fact, SHG microscopy highlights morphologic changes in collagen structure, which indicate particular disease states, such as tumour invasiveness, as well as indicators of collagen remodelling in tumour stroma, which is playing a key-role in the tumour development from in-situ to invasive stage.…”

From nano to macro: Studying the hierarchical structure of the corneal extracellular matrix

“…Over the past decade, NLO SHG imaging has been used to evaluate corneal collagen in normal corneas from human (Aptel et al, 2010; Han et al, 2005; Morishige et al, 2006), mouse (Lo et al, 2006), pig (Jay et al, 2008; Teng et al, 2006; Wang et al, 2008), and rabbit (Morishige et al, 2006), as well as used to study pathologic conditions, such as keratoconus (Morishige et al, 2007; Tan et al, 2006), intrastromal laser ablation (Han et al, 2004; Wang and Halbhuber, 2006), thermal injury (Lo et al, 2009; Tan et al, 2005), transgenic mouse models (Lyubovitsky et al, 2006), infectious keratitis (Tan et al, 2007), wound healing (Farid et al, 2008; Nien et al, 2011; Teng et al, 2007), corneal edema (Hsueh et al, 2009; Wu and Yeh, 2008), collagen crosslinking (Bueno et al, 2011), and diabetes (Latour et al, 2012b). More recently, objective measures have been developed to characterize collagen orientation by using fast Fourier transformation (Ghazaryan et al, 2013; Lau et al, 2012; Lo et al, 2012; Mega et al, 2012; Rao et al, 2009; Tan et al, 2013) and by taking advantage of the unique polarization dependent properties of SHG signals (Latour et al, 2012a; Stoller et al, 2002; Tuer et al, 2012).…”