Fig. 1.

Representative two-photon excitation fluorescence (TPF) and second-harmonic generation (SHG) images of ex vivo formalin-fixed, deparaffinized, unstained PXE-affected skin sections. a Green (525/50 nm) emission filters were chosen to detect a high portion of TPEF signals originating from elastin and calcification in the dermis. SHG signal of collagen was spectrally differentiated with the use of a narrowband 405/20 nm filter, displayed by magenta color. Excitation wavelength was 805 nm. It should be noted that keratin also emitted TPEF signal in the green channel. Individual TPEF and SHG images of 420 × 420 μm² were assembled into a two-color mosaic image. The mid-dermis contained confluent calcified areas and fragmented, clumped elastin fibers revealed by the TPEF channel. We described the same findings, when we previously compared TPEF and SHG image of PXE-affected and healthy skin [19]. b-c Representative histology images with tenfold magnification of the mid-dermis from the same skin sample. b von Kossa revealed mid-dermal calcium deposition and calcified elastic fibers. c Weigert’s elastic staining displayed fragmented and polymorphic elastic fibers. White arrows, elastin fibers; yellow arrows, calcium deposits; blue arrows, collagen fibers. Scale bars display 400 μm