# Dark-Field Hyperspectral Microscopy for Carbon Nanotubes Bioimaging
## 1 Introduction  
Since their first discovery in 1991 [1], carbon nanotubes (CNTs) have gained much attention from the various fields of science [2, 3, 4, 5, 6, 7] and industry [8, 9, 10] as a material with prominent physicochemical characteristics. Carbon nanotubes are the allotropic form of carbon, having a cylindrical hollow structure up to several tens of micrometres in length and, generally, up to tens of nanometres in diameter [11]. CNTs are formed by rolling up a graphene sheet, another allotropic form of carbon, consisting of hexagonally bonded atoms arranged in a honeycomb pattern. Nanotubes could be rolled up from either a single graphene sheet and thus named single-walled carbon nanotubes (SWCNTs) or multiple graphene sheets and hence named multi-walled carbon nanotubes (MWCNTs) [12]. The unique morphology and size of CNTs provide excellent mechanical properties, large specific surface area, high electrical and thermal conductivity, flexible surface chemistry and valuable optical properties [13]. These features have prompted CNTs to be applied as the key component in various composites, sensors, coatings, and devices [11, 14]. Moreover, hollow-tube-shaped structures coupled with the possibility of functionalisation make them highly engaging in biomedical applications [15]. For example, CNTs can be applied as radio-imaging probes in positron emission tomography (PET) and single-photon emission computed tomography (SPECT) [16, 17, 18], contrast agents in magnetic resonance imaging (MRI) [19] and Raman spectroscopy [20]. Strong optical resonances in the near-infrared region also allow the use CNTs in near-infrared and photoacoustic imaging techniques [21, 22, 23, 24]. Carbon nanotubes could also be adapted to modulate neuronal growth as biocompatible scaffolds controlling cell adhesion, differentiation and migration [25, 26, 27]. The distinctive shape of the nanomaterial favours building the biocompatible drug-delivery system for cancer or gene therapy [9]. However, despite where CNTs could be effectively applied, their extensive toxicity and biodistribution analyses are required [15]. It is undoubted that physicochemical properties significantly determine the toxicity of nanomaterials. In terms of nanotubes, the importantparameters, which form the fibre pathogenicity paradigm (FPP), are length, diameter, rigidity, and biopersistence [28]. The long and thin CNTs fulfil all FPP attributes and hence may possess highly inflammogenic and fibrogenic potential [29; 30]. Among different techniques, such as particle size analysis, X-ray diffraction analysis (XRD), mass spectrometry, infrared spectroscopy, electrophoresis, cytotoxicity, and viability assays, primarily used to define nanoparticles' chemical structure, size distribution or toxicity, the microscopy techniques stand out as the versatile tool relevant to morphology characterisation and toxicity analysis [31; 32; 33]. Nowadays, three principal approaches to the microscopy of CNTs can be highlighted: electron microscopy, scanning probe microscopy (SPM), and optical microscopy. Electron-based imaging systems as well as SPM methods, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM) or scanning tunnelling microscopy (STM), are primarily used to define the ultrastructure of fabricated nanotubes and their mechanical properties, such as accurate dimensions, the number of layers, interlayer spacing, surface functionalisation, Young's modulus, adhesion, and stiffness [34]. However, they are not suitable for dynamic bioimaging studies of nanotubes since such visualisation is either labour- and time-consuming or requires special conditions, invasive sampling, and expensive equipment. On the other hand, conventional optical microscopy lacks these drawbacks. Nevertheless, optical imaging also could be challenging for tubes with several nanometres in diameter due to the diffraction barrier caused by the wave nature of light.