Light Sheet Fluorescent Microscopy and Automated 3D Image Analysis for Quantitative Lineage Tracing in Mouse Cornea

Light sheet fluorescent microscopy (LSFM) can be used to obtain large-scale (up to whole organ) 3D images; however, once one turns to lineage tracing, there is a conflict between efficacy of tissue clearing, permeabilization for immunolabeling, and tracing signal salvation. We report a methodological and analytical pipeline that would aid researchers using multi-color (Confetti) lineage tracing and utilize LSFM along with immunolabeling procedures. We used mouse cornea as a primary object and provided its detailed characteristics at a cellular level using dual-view inverted selective plane illumination (diSPIM) microscopy. We developed a protocol for a 3D image acquisition and analysis pipeline of lineage tracing data in Prrx1-expressing cells of corneal stromal and endothelial compartments. Morphometry data included calculation of cell number in epithelium, stroma, and endothelium along with measurement of corneal thickness and radius of curvature. The developed image analysis pipeline allowed calculation of Confetti marker distribution and quantitative clonal expansion estimation with adjustment imposed to correct spectral Confetti channel leakages. We believe that the presented methodology and 3D image analysis scripts should facilitate the lineage tracing studies and may be adapted to other organs.