# Emerging Optical Techniques for the Diagnosis of Onychomycosis
## 2 Spectroscopy  
### Raman Spectroscopy (RS)  
Raman spectroscopy is an optical technique based on the inelastic light scattering resulting from molecular vibrations. Raman scattering is a two-photon event and involves the change in the polarizability of the molecule in relation to its vibrational energy. The incident photon exchanges energy with the molecule, creating an induced dipole moment. The radiation emitted by this induced dipole moment results in the observed Raman scattering. Distinct and chemical composition-specific bands in the Raman spectrum represent a so-called \"molecular fingerprint\" of the sample under study. RS of bioprobes provides ample physico-chemical information to allow for detailed analysis of tissue composition at molecular level, such as the investigation of human hard (hair and nails) and soft (stratum comeum, callus) keratins [16]. With minimal water interference RS is a non-invasive technique suitable for in vivo studies and has been successfully applied in bio-medical research, e.g., for the in situ diagnosis of malignancies [17]. However, it is limited by the laser-induced fluorescence of organic molecules and an overall low signal-to-noise ratio [18]. The cleavage of disulphide bonds is an essential step in the pathophysiology of onychomycosis, and therefore of central interest as a target Raman spectra profile for the identification of the infection. The intensities and the Raman shift of the Ca-C\(\beta\)-S-S'-C'\(\beta\)-C'\(\alpha\) band imply the conformation of the disulphide bridges. Cysteine residues appear at 450-590 cm\({}^{-1}\) and 600-650 cm\({}^{-1}\), assigned to the S-S stretching mode and C-S stretching mode, respectively (Figure 1). [PERSON] et al., [19] found a significantly lower ratio of peak areas of S-S to C-C bonds for onychomycotic compared to healthy nails, underlining the cleavage of the disulphide bridges. Moreover, a weak Raman peak at 2600 cm\({}^{-1}\), attributed to the S-H bond, was observed in diseased nails, further indicating the S-S bond break and the presence of the S-H moieties albeit in a lower proportion. On the contrary, [PERSON] et al., [20] did not observe significant alterations of the S-S band in Raman spectra of onychomycosis nails. Moreover, they could not identify the S-H peak in either healthy or diseased nails. This vast discrepancy in the findings of the two groups may be, at least partly, attributed to differences in the methodological steps of the two studies, including dissimilar pre-processing (baseline correction) of the Raman spectra, different laser excitation wavelength, integration times, photothermal effects, sample storing temperature, etc. For example, the viability of the fungi is maintained in samples stored at 4 \({}^{\circ}\)C even though further growth, i.e., continuing keratin degradation, is inhibited [6]. Structural changes of nail keratin before and after treatment of onychomycosis with a neodymium-doped yttrium aluminium garnet (Nd:YAG) laser were also explored by Raman spectroscopy focusing on the impact on the disulphide band [21]. The effect of laser treatment on nail samples with onychomycosis was evaluated as a significant reduction in intensity and area of the disulphide band, by 55% and 38% respectively. Deconvolution of the disulphide band into two peaks assigned to gauche-gauche-gauche conformation (506 cm\({}^{-1}\)) and the less stable gauche-gauche-trans conformation (518 cm\({}^{-1}\)) [22] indicated destabilization of the S-S bond of the infected keratin structure after laser treatment. Previous RS studies on onychomycosis nail clippings [23] suggested that in healthy specimens the disulphide bond adopts the energetically more favorable gauche-gauche-gauche conformation, while in onychomycotic nail S-S Raman stretching modes  
Figure 1: Raman spectra of nail clippings in the fingerprint region (400–1800 cm\({}^{-1}\)); healthy (black), infected with _T. rubrum_ (red). The spectra are vertically offset for clarity.