Low concentration Phloxine B staining for high chemical contrast, nonlinear microscope mosaic imaging of skin alterations in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is an autosomal recessive metabolic disorder characterized by ectopic mineralization of soft connective tissue. Histopathology findings include fragmented, mineralized elastic fibers and calcium deposits in the mid-dermis. Nonlinear microscopy (NLM) can be used for visualization of these histopathological alterations of the mid-dermis in PXE-affected skin sections. Upon introducing a normalized 3D color vector representation of emission spectra of three of the main tissue components (collagen, elastin and calcification) we found that due to their broad, overlapping emission spectra, spectral separation of emission from elastin and calcification is practically impossible in fresh-frozen or unstained, deparaffinized PXE sections. However, we found that the application of a low concentration Phloxine B staining after the deparaffinization process creates an imaging contrast for these two tissue components, which enables spectral decomposition of their fluorescence images. The obtained concentration maps for calcium deposits can be well suited for the determination of illness severity by quantitative analysis.