Abstract
BackgroundSecond Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet’s membrane, in the posterior cornea.Methodology/Principal FindingsWe studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet’s membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats.Conclusions/SignificanceOur study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.
Highlights
Multiphoton microscopy is a well-established optical technique in biology because of its capability for three-dimensional (3D) multimodal imaging [1,2]
Our demonstration of in vivo second harmonic generation (SHG) imaging opens the way to long-term dynamical studies. This method should be generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies
This region was readily located below the stroma that is characterized by striated SHG images and above the endothelium that shows low 2PEF signal from endothelium cells [9]
Summary
Multiphoton microscopy is a well-established optical technique in biology because of its capability for three-dimensional (3D) multimodal imaging [1,2]. SHG microscopy provides contrasted and specific images of the corneal stroma that is mainly composed of type I collagen fibrils organized in stacked lamellae and appears as a well-suited technique for cornea imaging [6,7,8,9]. This technique has been rarely used for the study of corneal pathologies, except the keratoconus [10,11]. This study aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet’s membrane, in the posterior cornea
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