A quantitative approach to histopathological dissection of elastin‐related disorders using multiphoton microscopy

Background Multiphoton microscopy (MPM) is a novel imaging technology that has recently become applicable for diagnostic purposes. The use of (near) infrared light in MPM allows for deep tissue imaging. In addition, this modality exploits the autofluorescent nature of extracellular matrix fibres within the skin. Objectives To quantitate the structure and abundance of elastic fibres in human dermis in three dimensions utilizing autofluorescent signals generated by MPM for the objective examination of elastin-related skin disorders. Methods Cross-sections of skin samples from elastin-related disorders were analysed by MPM and correlated to histopathology. In situ visualization of elastic fibres by MPM was conducted by en face imaging of ex vivo skin samples through the intact epidermis. Image analysis software was used to quantify elastic fibres in three dimensions. Results Based on the MPM-detected elastin-specific autofluorescence, we developed the Dermal Elastin Morphology Index (DEMI), calculated as the ratio of elastic fibre surface area and volume. This enabled objective three-dimensional quantification of elastic fibres. Quantitative scoring of sun-damaged skin using DEMI correlated with qualitative histopathological grading of the severity of solar elastosis. Furthermore, this approach was applied to changes in elastic fibre architecture in other disorders, such as pseudoxanthoma elasticum (PXE), PXE-like syndrome, elastofibroma, focal dermal elastosis, anetoderma, mid-dermal elastolysis and striae distensae. We imaged elastic fibres in intact ex vivo skin imaged en face through the epidermis, indicating that this approach could be used in vivo. Conclusions MPM has the potential for noninvasive in vivo visualization of elastic fibres in the dermis with near histological resolution. DEMI allows objective assessment of elastic fibres to support diagnosis and monitoring of disease progress or therapy of elastin-related skin disorders. What's already known about this topic? Multiphoton microscopy has been used on tissue sections and in patients to assess skin pathologies. Autofluorescent structures within the skin, including elastic fibres, can be visualized using multiphoton imaging without the need for histochemical or antibody staining. What does this study add? Development of the Dermal Elastin Morphology Index (DEMI) to enable objective three-dimensional quantification of elastic fibres. Use of DEMI is demonstrated in elastin-related skin disorders. DEMI can be used on tissue sections, where it correlates with histopathology, but in contrast to histopathology it allows in vivo application.