Laser Scanning Confocal Microscopy: Application in Manufacturing and Research of Corneal Stem Cells

Laser scanning confocal microscopes (LSCMs) are powerful devices used to acquire high definition optical images by choosing the required depth selectively. The presence of specific laser beams and features such as fluorescence recovery after photobleaching (FRAP), fluores‐ cence lifetime imaging microscopy (FLIM), and fluorescence resonance energy transfer (FRET) allow to: i. increase the quality of the image; ii. observe and analyze subcellular organelles; iii. track the localization of any given labeled molecule within the cell; iv. identify specific areas within a tissue/organ (Figure 1). In parallel, the development and manufacturing of fluorescent probes (=fluorophores) characterized by low toxicity profiles are allowing to perform the above mentioned studies using living cell cultures or tissues that are not fixed. Furthermore, fluorescent proteins such as the Green Fluorescent Protein (GFP) and its derivatives allow to detect how the biosynthetic machinery of the cell works or a transgene (driven by a plasmid or a genetically engineered virus) is expressed (Figure 2) or a chimeric protein interacts with other cellular components. The aim of this chapter is therefore to describe how LSCM functions and features have helped vision sciences and regenerative medicine applications in the field of ophthalmology. The next sections will analyze how LSCM-based analyses have helped to: 1. evaluate how the ocular surface is formed; 2. define the role of p63 as stem cell marker; 3. set up quality control assays required for clinical applications of limbal stem cells in patients with limbal stem cell deficiency (LSCD); 4. validate the use of impression citology as a diagnostic tool for LSCD; 5. study gene therapy-based potential ways to treat rare genetic disorders of the ocular surface.