Background: Ex vivo confocal laser scanning microscopy (ex vivo CLSM) provides rapid, high-resolution imaging of excised skin samples as well as a digital hematoxylin-eosin-like staining. Furthermore, immunofluorescence examinations can be performed with the combination of fluorescent-labelled antibodies.
Objective: To assess the applicability of ex vivo CLSM in the diagnosis of pemphigus vulgaris (PV) and to investigate the ideal staining protocol for direct immunofluorescence examination using ex vivo CLSM.
Methods: 300 µm thick sections of PV patients were incubated with different concentrations of IgG and C3 for various lengths of time and examined with ex vivo CLSM in reflectance and fluorescence as well as digital staining mode. 
Results: Ex vivo CLSM identified intraepidermal blister formation, acantholytic cells and subepidermal inflammation in 80%, 60% and 80% of PV patients, respectively. Immunostaining using fluorescent-labelled IgG with 1:20 and C3 with 1:80 dilutions enabled the ideal visualization of linear intraepidermal fluorescence in 90% of PV patients. The sensitivity of ex vivo CLSM in detecting intraepidermal fluorescence was 90% both with IgG and C3. The specificity of IgG and C3 was 70% and 90%, respectively. 
Conclusions: Ex vivo CLSM could detect histopathological features as well as intraepidermal fluorescence in probes from PV patients within the same session showing a comparable performance to conventional histopathology and direct immunofluorescence microscopy.

Simultaneous assessment of histopathology and direct immunofluorescence in pemphigus vulgaris using ex vivo confocal laser scanning microscopy