Micromechanical behavior of the apple fruit cuticle investigated by Brillouin light scattering microscopy

Abstract

The cuticle is a polymeric membrane covering all plant aerial organs of primary origin. It regulates water loss and defends against environmental stressors and pathogens. Despite its significance, understanding of the micro-mechanical properties of the cuticle (cuticular membrane; CM) remains limited. In this study, non-invasive Brillouin light scattering (BLS) spectroscopy was applied to probe the micro-mechanics of native CM, dewaxed CM (DCM), and isolated cutin matrix (CU) of mature apple fruit. The BLS signal arises from the photon interaction with thermally induced pressure waves and allows for imaging with mechanical contrast. The derived loss tangent showed significant differences with wax extraction from the CM and further with carbohydrate extraction from the DCM, consistent with tensile test results. Spatial heterogeneity between anticlinal and periclinal regions was observed by BLS microscopy of CM and DCM, but not in CU. The key conclusions are: (1) BLS is sensitive to micro-mechanical variations, particularly the strain-stiffening effect of the cutin framework, offering insights into the CM's micro-mechanical behavior and underlying chemical structures; (2) CM and DCM exhibit spatial micro-mechanical heterogeneity between periclinal and anticlinal regions.

Fig. 1. Mechanical characterization of the cuticular membrane.

a Averaged Brillouin spectra and standard deviation of the cuticular membrane (CM), dewaxed CM (DCM), and the acid resistant fraction of DCM (cutin matrix, CU) showed the water peak at around 7.5 GHz and the sample peak at around 15 GHz. Signal intensity was plotted logarithmically. Box plots of averaged Brillouin frequency shift (BFS; b) and Brillouin line width (BLW; c) of the extracted spectra peak of CM, DCM, and CU. Each sample point is the average over a 2D scan with 900 data points. d Force-strain diagrams of appropriately treated samples obtained from uniaxial standard tensile tests. One representative curve is shown in blue. * and ** denote p < 0.05 and p < 0.01 using the Mann-Whitney-U test, ns indicates non-significant differences.

Fig. 2. Thermal relaxation processes of the cuticular membrane.

Effect of heating and subsequent cooling of isolated and hydrated cuticular membrane (CM; a, b) and dewaxed CM (DCM; c) of ‘Idared’ apple on Brillouin frequency shift (BFS, average & standard deviation), Brillouin line width (BLW, average & standard deviation), and heat flow (differential scanning calorimetry; DSC) after a CM first temperature cycle and, b second temperature cycle. Additionally, the effect was investigated in DCM (c) after first temperature cycle. DSC thermographs show the melting temperature of the embedded wax. The distinct peaks in heat flow of CM wax (53 ± 1 °C, 61 ± 1 °C, and 67 ± 1 °C) are marked with the dotted lines. *, **, ***, **** denote p < 0.05, p < 0.01, p < 0.001, and p < 0.001 using mixed linear-effect model and post-hoc Tukey analysis.

Fig. 3. Spatial heterogeneities within the cuticular membrane visible with Brillouin light scattering.

a Brillouin intensity maps and the calculated masks for periclinal regions (PR) and anticlinal regions (AR) of the cuticular membrane (CM), dewaxed CM (DCM), and the acid-resistant fraction of DCM (cutin matrix, CU). Scale bars denote 20 µm. Representative normalized Brillouin frequency shift (BFS) b and Brillouin line width (BFS) c for AR and PR. Diagrams in b and c are scaled identically for CM, DCM, and CU, but have a different offset. Calculated ratio of averaged BFS d and BLW e for AR and PR. * and ** denotes p < 0.05 and p < 0.01 using the Mann-Whitney-U test.

Fig. 4. Schematic overview of the Brillouin light scattering micro-spectroscopy setup.

The light from the single longitudinal mode (SLM) laser was further filtered by a temperature-controlled etalon (TCE) before passing power regulation optics (λ/2-plate and polarized beamsplitter; PBS) and entering the microscope. The sample is mounted in a hydration chamber and gets analyzed with circular polarized light (λ/4-plate). The Brillouin-scattered light gets collected in back-scattering geometry and analyzed with the commercially available 3 + 3-pass Tandem-Fabry-Pérot interferometer TFP-2 HC featuring two pinholes (PH), an entry-pinhole camera (PHC) and a single photon avalanche diode (SPAD).