# Dark-Field Hyperspectral Microscopy for Carbon Nanotubes Bioimaging
## 4 In Vivo Studies of CNTs  
Dark-field microscopy is widely used to observe the distribution of CNTs in tissues and cells following in vivo exposure on animals. Table 2 summarises applications of DFM on animal tissue and cell extracts. Generally, for the microscopic examination of tissue samples, the examined organs are fixed with neutral buffered formalin (NBF), paraffin-embedded, sectioned, stained with histological dyes after sacrification at specified post-exposure time points. The staining of sections enhances the contrast between CNTs and the tissue, which is hard to distinguish due to low brightness. Notably, each study under investigation has applied CytoViva's enhanced dark-field imaging technique, increasing resolution compared to traditional DFM. For instance, [PERSON] et al. studied double-walled carbon nanotubes (DWCNTs) pulmonary bioactivity in the C57 BL/6 mouse model [82]. The particles appeared as bright tubular inclusions on the tissue processed from mice dosed with CNTs via pharyngeal aspiration 56 days post-exposure. The same features were also noted in tissue and cell samples of the other in vivo studies [83; 84; 85; 86]. Another study investigating the effect of surface carboxylation on CNT bioactivity showed that clearance of functionalised nanotubes from lungs occurred better than that of unmodified [87]. Similarly, the influence of carboxylation degree on CNT deposition in lungs of albino BALB/c mice was assessed by EDFM supplemented with hyperspectral imaging [88; 89]. However, the often-used Spectral Angle Mapper algorithm, detecting particles within biological specimens by comparing their spectra with the endmember spectrum, could not recognise CNTs in the samples due to the similarity of their spectrum to that of the background of stained tissue. Therefore, the Spectral Feature Fitting was applied, which compares sample spectra with the brightest spectrum of CNTs in tissues. The method showed that highly functionalised MWCNTs had a higher lung burden and were more dispersed (Figure 4A). They also appeared to associate more with epithelial cells rather than alveolar macrophages, as occurs with pristine and less functionalised nanotubes. In order to avoid potential artefacts caused by instillation/agglomeration of CNTs because of a high single dose of particles, inhalation studies that closely mimic environmental conditions are performed. For example, [PERSON] with colleagues have observed the toxicity of MWCNTs on Sprague Dawley (SD) rats after 5-day inhalation [90]. EDFM-HSI showed that nanotubes deposited in alveolar epithelium and macrophages persisted after 30 days post-exposure. The research group have pursued a study with extended exposure and postexposure times, finding that CNTs persist in the lungs after 90 days of the post-exposure period [91]. Another example of successful hyperspectral analysis is represented by the study of [PERSON] et al., where it was applied to detect SWCNT uptake into circulating cells of severe combined immunodeficient (SCID) mice [92]. EDFM-HSI provided a map of the subcellular spatial distribution of nanomaterial, suggesting internalisation rather than binding to the membrane.