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.

Studies on fixed samples or genome-wide analyses of nuclear processes are useful for generating snapshots of a cell population at a particular time point. However, these experimental approaches do not provide information at the single-cell level. Genome-wide studies cannot assess variability between individual cells that are cultured in vitro or originate from different pathological stages. Immunohistochemistry and immunofluorescence are fundamental experimental approaches in clinical laboratories and are also widely used in basic research. However, the fixation procedure may generate artifacts and prevents monitoring of the dynamics of nuclear processes. Therefore, live-cell imaging is critical for studying the kinetics of basic nuclear events, such as DNA replication, transcription, splicing, and DNA repair. This review is focused on the advanced microscopy analyses of the cells, with a particular focus on live cells. We note some methodological innovations and new options for microscope systems that can also be used to study tissue sections. Cornerstone methods for the biophysical research of living cells, such as fluorescence recovery after photobleaching and fluorescence resonance energy transfer, are also discussed, as are studies on the effects of radiation at the individual cellular level.