# Emerging Optical Techniques for the Diagnosis of Onychomycosis
## 3 Imaging Techniques
### Confocal Laser Scanning Microscopy (CLSM)  
CLSM is a complementary technique to conventional microscopy with increased resolution towards ultrastructural details. In conventional microscopy, the image quality may be seriously undermined by artefacts originating from specimen fixation and sectioning [40]. CLSM offers high-resolution images with a lateral resolution down to 140 nm and axial resolution of 1 mm, offering the visualization of the tissue in consecutive sections parallel to the surface of the sample. CLSM is a light reflection technique based on a spatial filter to block out-of-focus light. While light is illuminating the sample, a confocal microscope focuses a beam at one narrow depth level each time, achieving highly-confined depth of focus, effectively suppressing light from out-of-focus planes and reconstructing the image in x/y/z-directions [41].  
CLSM images of healthy fingernails reveal a three-zonal \"sandwich\" nail structure consisting of a brighter uppermost surface-reflection layer, followed by a layer with poorer signal and finally a brighter deeper zone (Figure 4). The nail bed is demonstrated as a wave structure, however, only in relatively thinner nails (\(<\)500 \(\upmu\)m) [42]. Single corneocytes can be visualized within the healthy nail plate while hyphae of dermatophytes were observed in onychomycotic nails [37]. Moreover, direct fungal quantification can be performed by analyzing the location and the density of the fungi [43].  
In vivo and in vitro confocal infrared microscopy, exploiting a 1064 nm Nd:YAG laser, revealed the presence of branched hyphae in both onychomycotic toenails of a patient [44]. [PERSON] et al., [45] examined 58 patients with clinical suspicion of distal and lateral subungal onychomycosis covering 10-75% of the surface of at least one great toenail by reflectance confocal microscopy (RCM). Onychomycosis was characterized by the presence of bright filamentous branching structures corresponding to hyphae on at least three consecutive images. RCM classified correctly 79.3% of the onychomycotic samples with 52.9% sensitivity and 90.2% specificity. Moreover, CLSM can detect the location and density of the pathogen but it cannot discriminate between dermatophytes and non-dermatophytic moulds or yeasts [46].  
Figure 4: Images of onychomycotic nail: (**a**) Clinical, (**b**) in vivo CLSM and (**c**) ex vivo CLSM. The hyper-reflective, branching, linear structures are typical of fungal hyphae. Reproduced with permission from [42].  
A detailed analysis of the performance of CLSM as diagnostic tool for onychomycosis was reported by [PERSON] et al. [PERSON] et al. (2018). In a prospective trial, 50 patients with suspected onychomycosis and 10 controls were cross-examined with CLSM in comparison to three reference techniques (PCR, fungal culture and PAS staining). CLSM showed the highest positive predictive value (88.6%) which was similar to KOH preparation (85.3%). The specificity of CLSM (81%) was superior to KOH preparation (76.2%) but inferior to PAS staining (100%). Moreover, CLSM (79.5%) exhibited a higher sensitivity to histopathology (69.2%) and KOH preparation (74.4%). Overall, although CLSM can principally aid the diagnosis of onychomycosis, it remains too sophisticated and expensive a technique to adopt for routine use in dermatological practice [PERSON] et al. (2018).