# Dark-Field Hyperspectral Microscopy for Carbon Nanotubes Bioimaging
## 3 In Vitro Studies of CNTs
It was discovered that carboxylation and base washing of MWCNTs increased cellular uptake. At the same time, coating nanotubes with bovine serum albumin (BSA) impacted cell absorption of only pristine nanotubes, which EDFM confirmed. Moreover, basic dye staining in a series of studies assessing the carcinogenic potential of pristine and surface functionalised CNTs on human small airway epithelial cells (SAECs) was applied to enhance cell contrast in EDFM [72; 73; 74]. Dark-field microscopy confirmed that carbon nanotubes either co-localised in the cytoplasm of cellsor punctured the cellular or nuclear membranes. Another study based on fluorescence and dark-field imaging showed that ovalbumin (OVA) could be successfully delivered into macrophages in the complex with CNTs [75]. [PERSON] et al. investigated the genotoxicity of raw, heat-treated (HT) and nitrogen-doped (ND) carbon nanotubes on BEAS-2B and SAECs [76]. Utilising fluorescence-enhanced dark-field imaging, they were able to quantitatively assess the nuclear uptake of the individual nanotubes, which revealed consistently higher partitioning of raw MWCNTs in nuclei of BEAS-2B cells (Figure 3B). Additionally, simultaneous incubation of cells with high doses of CNTs and gold nanoparticles (AuNP) demonstrated aggregation of particles on cells which has not been observed in TEM images, pointing to the complementary role of the methods in distribution studies [77]. Apart from direct observations, distinctive spectral features of carbon nanotubes allow for mapping them in cell samples via hyperspectral imaging. One study showed that pre-treatment of SAECs with CNTs significantly enhances viral infectivity [78]. HSI mapping successfully detected SWCNTs and pandemic influenza A H1N1 virus (IAV) in cell samples as well as changes in their distribution patterns during co-exposure, suggesting the presence of nanotubes may influence IAV behaviour (Figure 3D). In the comparative cytotoxicity analysis of clay and graphene-based nanomaterials, the hyperspectral imaging technique allowed the identification of nanoparticles in vital and paraformaldehyde (PFA) fixed A549 cells correctly [79]. It was found that graphene oxide nanosheets and MWCNTs are penetrating cells less than nanoclays at the same concentration. HSI alongside fluorescence microscopy was also used to visualise the uptake and fate of SWCNTs and MWCNTs in a study of the fibrogenic effect of carbon nanotubes on human lung fibroblasts (CRL-1490) [80]. Figure 3: (**A**) Dark-field image of RAW 264.7 macrophages exposed to MWCNTs (0.2 \(\mathrm{\SIUnitSymbolMicro g}\)/\(\mathrm{cm}^{2}\)) for 24 h. (**B**) Composite image of enhanced dark-field showing the MWCNT fibres in the white and blue fluorescent DAPI stained nuclei of BEAS-2B cells after 24 h of exposure. (**C**) Fluorescence-enhanced dark-field microscopy images of MRC-5 cells exposed to 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of MWCNTs for 24 h. Magenta colour shows F-actin (cytoskeleton), blue colour represents DNA (cell nuclei), and green colour  
shows MWCNTs. (**D**) Hyperspectral mapping of SAECs co-treated with SWCNTs (blue pixels) and pandemic H1N1 influenza A virus (red pixels). Figure 3A adapted from Ref. [64] under the terms of the Creative Commons Attribution License (CC-BY 3.0), scale not specified. Figure 3B adapted from Ref. [76] under the terms of the Creative Commons Attribution Attribution License (CC-BY 4.0). Figure 3C adapted from Ref. [67] under the terms of the Creative Commons Attribution License (CC-BY 4.0). Figure 3D adapted from Ref. [78] under the terms of the Creative Commons Attribution License (CC-BY 4.0), scale not specified.