Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation

Summary Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon—a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70–90 μm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti‐neuronal β‐III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20–30 μm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface. Lay description Human cornea is one of the most highly innervated tissues in the body. Sectioning of the cornea for the quantitative analysis of its innervation by immunohistochemistry is necessary during health and disease. Attempts to obtain frozen or paraffin‐sections of whole corneas parallel to the plane of its innervation are technically challenging and also time consuming due to the curvature of the tissue. In an effort to overcome this difficulty, here we report a contact‐free and rapid femtosecond laser based cutting method to obtain high quality surface parallel sections of the human cornea in less than 15 min. Furthermore, femtosecond based laser microtomy can be adopted easily for sectioning of all kind of biological samples including hard tissues (teeth and bone) and offers a more standardized way of tissue sectioning by avoiding the laborious processes of sample preparation and embedding thereby preventing the loss of valuable tissue sample.