(Invited) Long Term Intracellular Study of Carbon Nanomaterials at Ultra-Low Concentrations
Authors: Ignatova Tetyana , Jedlicka Sabrina , Rotkin Slava V. 
Published: February 2020

Given the importance of nanomaterials for medicine, health and biological sciences, a method for reliable intra‐cellular tracking of nanocarbon material at ultra‐low concentrations was investigated. Namely, a hyperspectral imaging of DNA‐functionalized single‐wall carbon nanotubes inside neural stem cells was demonstrated, over several mitosis cycles, also in 3D z‐stacking mode.
CoMoCAT SWCNTs wrapped by ssDNA GT(20) were introduced to C17.2 neural stem cells (NSCs), which are an immortalized embryonic murine stem cell line derived from the cerebellum. SWCNT concentrations ranged from 50 ng/mL to 5 ng/mL (0.11‐0.01 nM, based on the mass of an individual (6,5) SWCNT). These concentrations, which are up to 106 times smaller than in some earlier works.
Localization of SWCNTs introduced to C17.2 NSCs has been performed using Raman microscopy. Observing the Raman peak at 1588 cm‐
1, which is related to the SWCNT tangential G‐band allowed for clear imaging of the SWCNTs due to a very high signal‐to‐background ratio. The high spatial resolution of confocal Raman microscope allows one to resolve the SWCNT position in 3D. In Fig. 1 fast mapping of the cell area (panel a) allowed us to identify position of a nanotube (inside light blue rectangle area) as being close to the cell membrane. Panel b shows the high‐resolution G‐band image of the individual (11,0) SWCNT (identified by its RBM mode). Panels d-g in Fig. 1 show a sequence of Raman maps taken at varying z‐location. It is clearly seen that a center of hotspot associated with the nanotube moves, coming in and out of focus at different depths inside the cell. Similar images were produced for several samples with cells incubated with SWCNTs maintained for at least one (and up to 4) division cycles. In all specimens, SWCNTs are localized inside of the C17.2 NSCs, demonstrating further the need for longer term tracking and analysis of SWCNT effects on cells.
In conclusion, imaging of individual SWCNTs inside C17.2 NSCs has been demonstrated using confocal scanning Raman microscopy. Although the full amount of information contained in hyperspectral data exceeds typical experimental needs, the capability to correlate the cell and the nanotube images is critical to understanding the mechanism of long‐term action (up to 72 hr) of nanomaterials applied to the cell in ultra‐low doses (below 0.1 nM in this work). Our long‐term study confirmed that SWCNTs are retained in C17.2 cells for at least 72 hours, after 4 cycles of cell division. Finally, confocal Raman microscopy not only enabled a reproducible detection of individual SWCNTs, it allowed us to identify the SWCNT chirality, which demonstrates a potential for multicolor/hyperspectral imaging and tracking of single nanotubes in even longer‐term experiments on live cells.

Acknowledgements

Authors acknowledge use of shared Raman facilities supported by LU CREF grant. SSJ acknowledges support by NSF:CBET (IDR‐1014957) and FIG grant of LU. SVR acknowledges support by NSF (ECCS‐1509786). SSJ and SVR acknowledge LU CORE grant.

Figure 1