In BLS spectroscopy the incident photon undergoes inelastic scattering with thermally induced density waves. The Doppler shift induced by this process is proportional to the acoustic wave speed of the sample within the focal volume. This speed can be linked to the mechanical properties of the sample30,31. Despite its significance in translational research, its use in plant sciences remains marginal30,33.
In this study, BLS microscopy was applied to fully hydrated, enzymatically isolated apple fruit CM. Firstly, BLS signals from the native CM, DCM, and CU were recorded and validated with stress-strain measurements. Secondly, the influence of wax crystallization on the mechanical properties of the cutin-polysaccharide framework was investigated by heating the CM above the melting temperature of the wax to change crystalline wax into amorphous form. Thirdly, micro-mechanical topological heterogeneities within the CM originating from the periclinal and anticlinal cell walls were analyzed by Brillouin surface scans. This allowed to investigate the local impacts of the different CM constituents.
Cuticular wax is known to act as a filler in the cutin matrix. Wax fixes the elastic strain of the CM induced during fruit surface expansion16,17. Wax extraction from the CM results in a significant release of the elastic strain fixed by wax34. Additionally, wax extraction significantly decreases stiffness and fracture force of the CM17. This behavior was also observed in our study (Fig. 1, Table 1). BLS measurement of the CM and DCM revealed a significant decrease in BFS (Fig. 1b). This reduction in the BFS implies a decrease in acoustic wave speed and thus, in mechanical stiffness. This observation is in line with previous macro-mechanical studies17,22,35,36.
The treatment of the DCM with HCl extracted polysaccharides embedded in the cutin network35,36. Extracting the polysaccharides resulted in a further significant reduction in stiffness, fracture force, and also a significant reduction in BFS compared to DCM. (Fig. 1, Table 1). This finding underlines the critical role of polysaccharides in the mechanical properties of apple fruit CM. Comparable results were reported for tomato fruit CM following CHO extraction21. This also aligns with the findings of the macro-mechanical uniaxial tensile test, where the removal of load-bearing components typically results in decreased stiffness. For instance, in cellulose composites, cellulose fibers serve as the primary load-bearing component37.
It is anticipated that trends in stiffness (as determined by tensile testing) and BFS will be correlated. However, it is crucial to recognize that these mechanical properties cannot be directly quantitatively compared. This discrepancy arises from the fact that Brillouin spectroscopy probes the ratio of uniaxial stress to strain in a confined environment. Thus, allowing for a density and/or volume change31. In contrast, the uniaxial tensile test requires the volume to be kept constant. However, the observed reduction in BFS is noteworthy, particularly considering its magnitude (BFS = 0.9 GHz) compared to similar studies involving other plant samples. Previous research has demonstrated that even subtle BFS changes in plant samples can have pronounced effects on macro-mechanical behavior25,27.
In the heating and subsequently cooling experiments, the CM were subjected to temperature increases above the melting temperatures of the embedded wax (Fig. 2a, b), which were determined with DSC and cooled back to room temperature. This experimental approach induced a phase transition, reflected in the BLS signal and the DSC thermographs. The observed hysteresis in the BFS in the first heating and cooling cycle (Fig. 2a) can be attributed to the melting of the wax crystals and resulting relaxation of the cutin network during heating and the resolidification of wax in its amorphous form (without additional dilation of the cutin network) during cooling17. The transition was observed to be less pronounced in the BFS compared to BLS studies involving pure paraffin, as reported in the literature38–40. This is likely linked to the lack of a network-like polymeric structure as the cutin polysaccharide framework in these studies.
The second temperature cycle showed no hysteresis in the BLS signal and reduced heat flow, indicating that the wax remains amorphous in this short time frame. This finding is consistent with previous observations made using Fourier-domain infrared spectroscopy17. This underlines the importance of wax crystallinity for the strain fixation functions, likely via withstanding the lateral forces exposed to the CM. Heating the CM results in a loss of crystal structure of the embedded wax and a partial release of elastic strain17. Consequently, the effect may be considered analogous to wax removal, albeit to a lesser extent due to the residual filling effect visible in the BLW (Fig. 2b and c).
This prompts the hypothesis that the measured BLS signal predominantly captures the stiffness within the cutin-polysaccharide matrix, where the molecular chains of the cutin network exhibit a strain-stiffening effect. A similar strain-stiffening effect has been demonstrated using BLS spectroscopy for spider silk under external tensile load41,42.
The BFS is related to the longitudinal modulus (\(M\)) via the density of the sample (\(\rho )\), the laser incident wavelength (\(\lambda\)) and the refractive index (RI) (\(n\)) as in Eq. 131:
$${M=\rho \left(\frac{{\lambda \omega }_{B}}{2n}\right)}^{2}.$$
1
Accordingly, local changes in refractive index could impair the interpretation of the micro-mechanical behavior. It is crucial to highlight the absence of a significant change in RI within the cuticle, as observed via RI tomography for the extraction steps (Supplementary Fig. 7). Additionally, as the embedded wax is not water soluble, we assume for the temperature cycle experiments that for a given temperature both the density and the RI stayed constant.
This stability of RI suggests that the observed Brillouin signal variations in the extraction processes and upon temperature cycling are not driven by concurrent changes in the optical properties and thus, implies that the observed BFS changes can be directly linked to changes in the mechanical properties of the sample.
In both isolated CM and DCM, the lateral BLS scans revealed a consistent micro-mechanical pattern mirroring the imprints of previously (before isolation) underlying epidermal cells (Fig. 3a). In CM, a significantly reduced BFS was observed in anticlinal regions (AR) in comparison to periclinal regions (PR) (Fig. 3b, and 3d). This implies a significantly lower stiffness in the AR than in PR of the CM.
Heating of the CM did not affect the BFS ratio (Supplementary Fig. 6). From this it is evident that, while the wax undergoes a change in state from crystalline to amorphous (Fig. 2), there is no redistribution of the waxes. Thus, the mechanical spatial heterogeneity found in native CM are maintained (Fig. 3b).
In DCM, the AR exhibited a significantly higher stiffness compared to the PR. This reverses the AR/PR relationship observed in CM (Fig. 3d). The larger reduction of stiffness in PR implies that waxes contribute more to the stiffness of the PR than of the AR.
The DCM major constituents are cutin and embedded polysaccharides, mainly cellulose, hemicellulose, and pectin2,18,43. These emerge from the epidermal cell walls43, which dominate the mechanical properties, similar to the CM of tomato fruits21,37,44. Cellulose is composed of long, ribbon-like linear chains45. These molecular chains have an angular invariance of the elastic tensor, which has a higher component along the fiber46. Accordingly, a higher BFS is anticipated when measured along the fiber, as it has been shown in spider silk41,42 and bamboo fibers29. Therefore, the observed higher BFS in the AR suggests a potential difference in the orientation of the crystallized polysaccharides following the orientation in the cell walls47. Supporting this hypothesis, the mechanical spatial heterogeneity disappeared in CU, both for the BFS and the BLW (Fig. 3).
By verifying our results with tensile testing and evaluating the possible influence of the RI, this study established Brillouin spectroscopy as a novel, noninvasive, and potentially in-vivo capable methodology enabling the measurement of mechanical properties, marking an important step regarding a deeper investigation of cuticle micro-mechanics. The results show that the cuticle cannot be regarded as a mechanically homogeneous construct, but has a pronounced micro-mechanical structure, which potentially has implications for the overall stability of the CM and the occurrence of microscopic defects.