Noninvasive visualization of intraepidermal and subepidermal blisters in vesiculobullous skin disorders by in vivo reflectance confocal microscopy

IntroductionBullous dermatoses are characterized by skin blisteringresulting from local injury with breakdown of tissue integrityand fluid accumulation within specific layers of the skin.These disorders are traditionally classified into subcorneal,suprabasal, and subepidermal blistering by the specificlocationofthesplitintheepidermis[1, 2]. Of special interestis the group of autoimmune blistering diseases that comprisea wide spectrum of clinical presentations and are mediatedby pathogenic antibodies targeting specific adhesion mole-cules responsible for cutaneous homeostasis and integrity[3]. Diagnosis is based on the clinical picture, histology,direct and indirect immunofluorescence, immunoblotting,immunoprecipitation and immunoelectron microscopy [4, 5].Though crucial for accurate diagnosis and for selection ofspecific therapy, these techniques are cumbersome, time-consuming and unlikely to be widely available, leavingblister level determination by classical histology a keydiagnostic procedure in intraepidermal and subepidermalblistering diseases.Reflectance confocal microscopy (RCM) is a novel,noninvasive imaging technique which permits real timevisualization of cellular components in the skin at aresolution compatible with that of conventional histology[6]. RCM detects single back-scattered photons directlyfrom illuminated living tissue without prior preparation ofthe examined skin. A small pinhole in the front of thedetector allows imaging at high resolution. Contrast inconfocal images is provided by the differences in refractiveindex among the cellular organelles and structures. Melaninacts as a contrast agent in pigmented epithelia [7]. With thecurrent technology, in vivo RCM imaging is limited to adepth of approximately 300 µm which includes the entireepidermis, the papillary dermis and the upper reticulardermis. It is of interest that the depth of RCM imaging canbe increased by using the so-called “optical clearing”approach, and experimental studies utilizing gold nano-particles and osmotically active immersion liquids asoptical clearance agents have indeed increased the imagingdepth of RCM up to three times [8–11]. However, theseexperimental approaches have not yet been incorporatedinto clinically useful RCM technology. Since contrast in theimages is primarily provided by melanin, RCM has mainlybeen useful in the diagnosis of pigmented cutaneous tumorssuch as melanoma and nevi [12], and nonmelanoma skincancers [13, 14]. The use of RCM has also been reported inthe diagnosis of a variety of inflammatory skin disordersincluding psoriasis [15], contact dermatitis [16, 17], vitiligo[18], cutaneous lupus erythematosus [19, 20], folliculitis[21], and photoaging [22]. In a recent paper, Angelova-Fischer et al. [23] demonstrated the use of RCM in thediagnosis of subcorneal blisters in two patients withpemphigus foliaceus in which the dark nonrefractive blistercavity was readily visible against a background of bright