Real-time snapping dynamics and nanoscale thickness profiling of salmon keratocyte tunneling nanotubes using quantitative phase microscopy

The highly migratory skin epithelial cells of fish, named keratocytes, display interesting features, including long cellular protrusions resembling tunneling nanotubes (TNTs). It has been shown in other cell types that TNTs may transfer cellular cargo between cells. These TNTs are instrumental not only in cargo transport but also in mediating signaling between cells. Due to the varying height of TNTs, down to 100 nm or less, a highly sensitive quantitative microscopy technique is required for accurate quantification of their morphological and dynamic properties. In this study, a partially spatially coherent quantitative phase microscopy (QPM) system was utilized, providing extremely high spatial sensitivity to track changes in TNT height over time, particularly during the breaking/snapping process. A phase-shifting technique was employed to recover high-resolution phase maps of the TNTs. Observations indicate that TNT height typically ranges from 100 nm to 700 nm. Additionally, the average height-to-width ratio over time suggests that the TNTs adopt a less rounded but flattened shape. Comparisons between intact and broken TNTs further reveal that unbroken TNTs generally exhibit a higher average height, with a maximum observed value of approximately 900 nm. Beyond morphological characterization, this work may open new avenues for understanding TNTs´ potential role in the transfer of intracellular cargo, paving the way for future investigations into cellular communication mechanisms.