In this work, we investigated the labeling efficiency of hydrophilic (DAPI) and hydrophobic (Nile Red) fluorescent probes using uni- and bi-directional diffusion on porcine liver tissue sections on the order of a few hundred microns in thickness. Bi-directional diffusion significantly enhanced the penetration of probes. Although diffusion most accurately describes the labeling process, we found that after removal of the labeling molecules residual probe molecules in the tissue sections continued to be transported deeper into the tissue resulting in additional tissue constituents being labeled. Our study shows that bi-directional labeling can significantly enhance the labeling of thick tissues for applications in which structural information in three dimensions is needed.
