Background/aims Fibre-optic confocal imaging (FOCI) allows non-invasive visualization of live skin in vivo. A contrast agent, a fluorophore, is injected into the dermis. FOCI images are horizontal optical sections with cellular resolution. The aim was to study in vivo epidermal changes and the cellular structure of keratinocytes in moderate to severe atopic eczema (AE). Methods Eight patients with AE with active lesions on the forearms were studied and compared to a control group of six healthy individuals, and two cases of AE without activity. Fluorescein sodium was used as fluorophore. A hand-held fibre-optic laser scanner (Stratum (R)) was used. The study included morphometric analyses. Results The confocal in vivo images identified characteristic features of epidermis and keratinocytes in active AE vs healthy skin controls. FOCI could non-invasively image acanthosis, spongiosis, and parakeratosis in AE. Epidermal oedema and micro-vesicles were visualized. Morphometry based on FOCI demonstrated 14% increased width of keratinocytes of atopic skin vs healthy controls. The epidermal structures and organization in distinctive cell layers were deviant as a result of the disease. Conclusions Fibre-optic confocal imaging can visualize essential epidermal structures of atopic eczema directly in vivo, in real-time, and with cellular resolution thus without disturbing the natural state of the skin. FOCI is primarily a research tool, but with a potential to become used in the clinic for non-invasive microscopic diagnosis of AE and monitoring of effect of therapies.