# Dark-Field Hyperspectral Microscopy for Carbon Nanotubes Bioimaging
## 3 In Vitro Studies of CNTs  
Dark-field microscopy is a versatile and easy-to-use tool for label-free, non-invasive observation of nanomaterials in vitro using model cell cultures. At the moment, DFM has been actively applied to visualise organic, inorganic and composite nanomaterials [60; 61]. Carbon nanotubes possess a high aspect ratio and strong Rayleigh scattering, allowing for the detection of them in biological specimens. Table 1 summarises studies on mammalian cells treated with CNTs with the application of optical dark-field microscopy. Most of them involve the various CytoViva's enhanced dark-field microscopy setups since the unique illumination approach significantly increases signal-to-noise ratio resolving to observe even single nanotubes. For example, the synergistic effect of SWCNTs and etoposide on human pancreatic adenocarcinoma cells (PANC-1) viability were studied [62]. EDFM imaging demonstrated a time-dependent accumulation/distribution of SWCNTs in living cell samples, although it was hard to confirm CNT internalisation inside the cells. The same cell model was used in another study, where MWCNTs and polyethene glycol (PEG) composite was used for photothermal treatment of pancreatic cancer [63]. Using EDFM, dose-dependent internalisation of MWCNTs-PEG into PANC-1 cells after 1 h exposure was detected. An example of visualisation of single CNTs is reported in another toxicological study (Figure 3A) [64]. The murine macrophage cells (RAW 264.7) exposed with MWCNTs for different time points (2-48 h) were directly observed through EDFM, confirming the time-dependent accumulation of nanotubes onto the surface as well as inside the cells. While examining the influence of the biological environment on the dispersion of CNTs, the EDFM technique was applied to confirm the bioavailability of nanotube dispersions to immortalised bronchial epithelium cells (BEAS-2B cell line) [65]. The complications arising from identifying cellular compartments affected by CNTs could be overcome using correlative dark-field imaging of nanoparticles and fluorescence microscopy. Notably, this approach has been applied in a study focused on the repair mechanism of lung epithelial tissue in the presence of pollutants [66]. The CNT-treated human lung adenocarcinoma cells (A549 cell line) were used as the epithelial tissue model with inhaled particles to observe their influence on recovery kinetics. CytoViva dual-mode fluorescence-enhanced dark-field microscopy setup was used to obtain 3D-rendered images of cells stained with 4\({}^{\prime}\),6-diamidino-2-phenylindole (DAPI) and rhodamine-phalloidin, which revealed strong attachment of CNTs to the cellular membrane and partial internalisation. The same technique was used in the study of proinflammatory and profibrotic activity of MWCNTs in A549 cells, macrophages (differentiated THP-1 cells) and fibroblasts (MRC-5; Figure 3C), that demonstrated comparatively higher uptake of CNTs in macrophages upon acute and prolonged exposure [67]. It is noted that increased uptake of nanotubes by macrophages could be due to their phagocytic activity and may cause fibrosis, which is also confirmed in the toxicogenic analysis of macrophages treated with similar CNTs concentrations and exposure time [68]. Besides, a 3D rendering approach was applied to assess the internalisation of \"long and thick\" Mitsui-7 and \"short and thin\" Nanocyl-7000 MWCNTs on various cell types (bronchial and alveolar epithelial cells, mouse macrophages, and mesothelial cells) [69] as well as a complex organotypic model of alveolar tissue [70]. Nanocyl-7000 nanotubes appeared as densely packed aggregates in both cases, while Mitsui-7 were distinguishable as individual nanotubes. Nevertheless, the internalisation of both types of MWCNTs was observed in all cell models. Additionally, the cellular uptake of CNTs could be affected not only by dimensionality but also surface modification. [PERSON] and colleagues investigated the influence of CNT functionalisation and protein corona formation on their biocompatibility [71].