# Clinical Applications of In Vivo and Ex Vivo Confocal Microscopy
## 3 Ex Vivo Confocal Laser Scanning Microscopy
### Main Ex Vivo CLSM Clues of Skin Cancer and Inflammatory Skin Diseases  
As previously mentioned, the most employed application of FCM is represented by margin definition of BCCs during Mohs surgery. Ex vivo CLSM allows a quick evaluation of surgical skin samples, directly in the surgical room. Therefore, this technique represents a potential replacement of intraoperative histology for tumor margins assessment on frozen tissues during Mohs surgery. Mosaics are generally created and interpreted in 10 min, while the preparation of cryopreserved sections for histopathologic examination may require 20 to 45 min per excision, becoming time and cost consuming during daily clinical practice [7,8]. One of the limitations of ex vivo CLSM seems to be related to the poor ability to differentiate cytological and architectural details. Accordingly, a specific staining cannot distinguish between different cell types, although the use of fluorescent-labelled specific antibodies may overcome this problem [103, 104, 105]. Similarly to FCM, FCM shows areas of high fluorescence organized to form structures/aggregates in contrast with the surrounding hyporeflective stroma or cells agglomerate, corresponding to tumor islands, with peripheral palisading, in BCCs. These structures are surrounded by hypofluorescent dark-appearing space, corresponding to clefting which is typical of superficial and nodular BCCs and less demarcated in infiltrative ones [83,106]. Additionally, other BCC features are represented by nuclear pleomorphism visualized as a variation of nuclear shape and/or increased nucleus/cytoplasm ratio and modified stroma surrounding the BCC nests, showing a dark background with fluorescent dots and filaments that correspond to fibroblasts, collagen fibers, and inflammatory infiltrate [83,106]. [PERSON] et al. reported that FCM had an overall sensitivity of 88% and specificity of 99% in detecting residual BCC, on a sample of 69 BCCs [106]. Similar results were obtained in a prospective study by [PERSON] et al. on a sample of 753 BCCs [107]. FCM enables the identification of different features in diverse BCC subtypes, with proliferation of atypical basaloid cells forming an axis parallel to the epidermal surface and cleft-like spaces typically described in superficial BCCs and nodules with peripheral palisading and clefting in nodular BCCs (Figure 3c,d). Infiltrative BCC is the most challenging subtype, formed by columns and cords of basaloid cells mixed with a collagenic stroma [83,84,108]. FCM has also been applied to margin assessment in SCC [109; 110]. A total of 41 out of 43 margins in 13 SCC were positive in both FCM and histopathology, therefore providing a good correlation between the two evaluations. In detail, confocal criteria for the diagnosis and evaluation of the degree of differentiation have been described, such as well-defined tumor silhouette, presence of numerous low fluorescent keratin pearls, keratin formation corresponding to fluorescent scales in well differentiated SCC and marked nuclear pleomorphism in poorly differentiated tumors [109]. Interestingly, a recent experience of margin assessment in LM/LMM with ex vivo CLSM has been reported in the literature, demonstrating a good rate of correct margins identification for LM/LMM (95.2%). In this study, the authors combined in vivo preoperative analysis an ex vivo one, a promising approach for mucosal melanoma, LM and LMM, especially when aesthetically or functional relevant areas are involved, in order to avoid staged excision [87]. A preliminary study on inflammatory skin diseases with FCM was performed by [PERSON] et al. with the aim to verify the correspondence between histologic images and confocal ones. Despite limitations related to manual sectioning of the specimen, this study showed an encouraging percentage of correct diagnosis. Psoriasis, in particular had the highest reliability (90.5%), while eczema showed the least reliability [99]. Recently, [PERSON] et al.