Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare disorder caused by mutations of the ABCC6 gene in which decreased plasma levels of inorganic pyrophosphate (PPi) lead to ectopic mineralization resulting in severe dermatological, ophthalmologic and cardiovascular complications. Components of the dermal connective tissue can be differentiated with nonlinear optical (NLO) techniques, as collagen emits a strong second harmonic generation (SHG) signal given its non-centrosymmetric structure, while elastin as an endogenous chromophore can be visualized by two-photon excitation fluorescence (TPF). We carried out deep phenotyping, molecular genetic testing and plasma PPi measurement of five patients with PXE. Ex vivo SHG and TPF imaging and conventional histopathological evaluation of their skin was performed and compared to five age- and gender-matched healthy controls. Pathological mutations were found in the ABCC6 gene in all patients and their plasma PPi levels were significantly lower compared to controls by unpaired t test (p=0,0005). We visualized and quantified the degradation and mineralization of elastin fibers and extensive mid-dermal calcium salt deposition as well as the alterations of the collagen structure in the mid-dermis of patients with PXE utilizing TPF and SHG, respectively. Based on our data, NLO enables high-detail imaging and quantification of the specific features of PXE-affected skin. In vivo NLO may be a promising method in the assessment of PXE in the future, facilitating early diagnosis and monitoring.

Nonlinear microscopy for the visualization of calcification and assessment of connective tissue fibers in pseudoxanthoma elasticum