Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum

Abstract

Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up.

Keywords: Calcification; Elastin; Multiphoton microscopy; Nonlinear optical microscopy; Pseudoxanthoma elasticum.

Fig. 1

Plasma inorganic pyrophosphate (PPi) levels of our PXE patients compared with age- and gender-matched healthy controls. Blood samples were collected and platelet-free plasma was prepared. a Plasma PPi levels of all included patients were determined by first converting PPi to ATP, then measuring ATP content in a bioluminescent assay. PPi concentration of plasma was calculated based on calibration standards and corrected for initial plasma ATP concentration. b Plasma PPi levels in PXE vs control patients (mean ± SD 0.232 ± 0.063 μM vs 0.947 ± 0.108 μM) were compared using unpaired Student’s t test. *p < 0.05. Pt, patient; Co, control

Fig. 2

Representative histology images of the mid-dermis of healthy and PXE-affected skin, stained with hematoxylin and eosin (H&E), Weigert’s elastic (WE), von Kossa (VK), and Van Gieson’s (VG) staining. Red arrows, fibroblasts; black arrows, elastic fibers. Scale bars display 200 μm

Fig. 3

Two-photon excitation fluorescence (TPF) and second-harmonic generation (SHG) images of PXE-affected and healthy skin. TPF signal (shown in green color) is mainly emitted by elastin in the dermis, while SHG (magenta) displays the collagen structure, both generated at 800-nm excitation wavelength. 420 × 420 μm² frames were captured and assembled into (a, b) mosaic images of skin samples with 300 × 300 μm² high zoom images of the dermis (α, β, γ, δ). a Control skin, b skin of patient 3. High zoom inserts were marked with (c–j). Representative high zoom images of the mid-dermis, c, d patient 1, e, f patient 2, g, h patient 4, i, j patient 5. White arrows, elastin fibers; blue arrows, collagen fibers; yellow arrows, calcium deposits. Scale bars display 500 μm for mosaic images and 100 μm for high zoom images

Fig. 4

Extent of calcification and amount of elastin fibers in two-photon excitation fluorescence (TPF) images of the dermis of PXE patients and healthy, and results of CT-FIRE analysis of second-harmonic generation (SHG) images of collagen in the dermis of PXE-affected and control skin. a Number of elastin fibers in PXE vs control patients (mean ± SD 175.7 ± 79.49 μm vs 106.8 ± 23.92 μm), b length of elastin fibers in PXE vs control patients (59.49 ± 29.9 μm vs 91.64 ± 74.72 μm), c relative calcification surface area in each PXE patient. Right panels show representative TPF images of PXE for each parameter. Statistical analyses were carried out using Mann-Whitney U test. e, f Collagen fiber length (mean ± SD 67.99 ± 3.612 μm vs 73.53 ± 4.782 μm) (e) and width (6.797 ± 0.274 μm vs 7.327 ± 0.325 μm) were compared using Student’s t test after customized CT-FIRE analysis of raw SHG images. Representative images of CT-FIRE v.13 (LOCI, University of Wisconsin – Madison, WI, USA) analysis of patient 1 and a healthy control. Scale bars display 100 μm. *p < 0.05