Confocal microscopy with in vivo and ex vivo modalities has been used in the evaluation of skin cancer and other dermatological disorders. Recent developments in ex vivo confocal microscopy allow for faster pathology assessment with greater accuracy by the visualization of cellular and architectural details, similarly to standard pathology, in either paraffin‐embedded or frozen samples. They include the possibility of multimodal confocal microscopy using different lasers and fusion images. New staining protocols including immunostaining, with no damage to conventional histopathology preparation, have been recently described in melanocytic tumours and inflammatory skin diseases. Digital staining with haematoxylin and eosin is also incorporated in the new devices. In this review the applications of ex vivo confocal microscopy will be presented with the description of the technique and the technology, clinical evidence in dermatology and other fields, and further applications.
For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4 × histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.
