Structural imaging of keratoconic human corneas using polarization-resolved Second Harmonic Generation microscopy

Multiphoton microscopy has revolutionized three-dimensional (3D) imaging of biological tissues by offering new modes of contrast even in unstained tissues. Notably, second harmonic generation (SHG) microscopy enables the visualization of fibrillar collagen without any labelling and with unequalled sensitivity and specificity [1] . Collagen is the most abundant protein in mammals and the main component of connective tissues, such as arteries, skin, bone or cornea. The size and the three-dimensional (3D) distribution of collagen fibrils are key distinctive features of every tissue that are crucial for its functional behaviour, notably its mechanical properties. Any disruption of this 3D structure may result in a pathological dysfunction of the tissue. In this context, polarization-resolved SHG (P-SHG) microscopy has been shown to provide a more accurate characterization of the 3D organization of collagen [2] , [3] because the SHG signal is higher when the excitation electric field is parallel to the dipoles accounting for the nonlinear optical response. The acquisition of a series of SHG images recorded with linear incident polarizations of various orientations thus provides the orientation of collagen fibrils in every pixel, using appropriate data processing based on a tensorial analysis of collagen response. This advanced multiphoton modality has been used in many tissues, including cornea that is composed of thin collagen fibrils aligned within 1–3 µm thick lamellae superimposed along the depth of the cornea, parallel to its surface [2] .