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

Fish skin epithelial cells, known as keratocytes, are highly migratory and exhibit distinctive morphological features, including elongated membrane protrusions called tunneling nanotubes (TNTs). In other cell types, TNTs have been reported to facilitate the intercellular transfer of various cellular cargo. Beyond cargo transport, these structures are also implicated in mediating intercellular signaling. Owing to their extremely small heights, often as low as 100 nm or less, reliable characterization of their morphology and dynamics necessitates the use of a highly sensitive quantitative phase microscopy technique. 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 snapping process. A five-frame phase-shifting technique was employed to recover high-resolution phase maps of the TNTs. Observations indicate that TNT height typically ranges from 100 to 900 nm. Additionally, the average height-to-width ratio indicates that the TNTs exhibit a more flattened rather than cylindrical geometry. Further, during the imaging period, diverse behaviors were observed: some TNTs remained intact and maintained structural integrity for the entire duration of the recording, whereas others exhibited rupture. Beyond morphological characterization, this work may open new avenues for understanding TNT’s potential role in the transfer of intracellular cargo, paving the way for future investigations into cellular communication mechanisms.