Figure 2. Gold standard alignment of intravital multiphoton tomography.

(a) Standard vertical histological sections of the skin from healthy subjects (healthy), the ostensibly healthy skin of a patient affected by AD (AD non-lesional) and lesional atopic skin (AD lesional) examined via multiphoton tomography. Alignment of horizontal histological sections (‘hist.’-column) with multiphoton tomographic sections (‘MPT’-column) of the same regions of healthy, AD non-lesional and AD lesional areas of skin at distinct skin depths of 0 μm (Stratum corneum (b)), 10 μm (upper Stratum granulosum (c)), 30 μm (Stratum spinosum (d)) and 50 μm (e), representing the interface between the epidermal Stratum basale and the Stratum papillare of the dermis. A comparison of ‘hist.’ with ‘MPT’ illustrates the identical validity of assessing the morphological skin architecture and detecting characteristic skin morphologies. In the ‘AD lesional’ patient, the dermis is not accessible at a depth of 50 μm due to typical thickening of the epidermis. The accessibility of the dermis was demonstrated through additional imaging of the collagen-specific generation of second harmonics, as shown in the backmost image of the composite ((e) ‘MPT healthy’ and ‘MPT AD non-lesional’). Furthermore, the keratinocytes appear to be dispersed by inflammatory intercellular oedema. At a depth of 30 μm ((d) ‘AD lesional’), MPT is conspicuously superiour to the histological preparation regarding the assessment of the intensity of intercellular oedema (marked with a blue box). Scale bars correspond to 20 μm.