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Three- and four-dimensional imaging techniques are a prerequisite for spatially resolving the form–structure–function relationships in plants. However, choosing the right imaging method is a difficult and time-consuming process as the imaging principles, advantages and limitations, as well as the appropriate fields of application first need to be compared. The present study aims to provide an overview of three imaging methods that allow for imaging opaque, large and thick (>5 mm, up to several centimeters), hierarchically organized plant samples that can have complex geometries. We compare light microscopy of serial thin sections followed by 3D reconstruction (LMTS3D) as an optical imaging technique, micro-computed tomography (μ-CT) based on ionizing radiation, and magnetic resonance imaging (MRI) which uses the natural magnetic properties of a sample for image acquisition. We discuss the most important imaging principles, advantages, and limitations, and suggest fields of application for each imaging technique (LMTS, μ-CT, and MRI) with regard to static (at a given time; 3D) and dynamic (at different time points; quasi 4D) structural and functional plant imaging.

[This corrects the article DOI: 10.3389/fpls.2025.1572745.].

Deep understanding of the structural composition and growth of biological specimens is becoming increasingly important for the development of bio‐based and sustainable material systems. Full‐field nano‐computed tomography is particularly suitable for this purpose as it allows for non‐destructive 3D imaging at high spatial resolution. However, most biological samples are functionalized by water and respond sensitively to any changes in climate conditions, specifically relative humidity, by adjusting their material moisture content. To date, only a limited number of tomography instruments offer an in situ climate control option to users. These, however, are limited either by the range of relative humidity states, the long times required to change the climate state, or obstruction or attenuation of the beam. Here, the first fully automatized climate cell for in situ full‐field nanotomography is presented. It has been designed, built and integrated at the nanotomography station at the P05 imaging beamline, operated by Hereon at the DESY storage ring PETRA III, Germany. The highly flexible and windowless design allows the humidity dependent swelling and shrinking of lignified plant cell walls to be studied in situ, using phase contrast nanotomography. The concept of this climate chamber can easily be integrated into other setups. It operates in the relative humidity range of 0–90% and can be controlled in a temperature range of 10–50°C. Climate conditions can be adjusted at any time, remotely from the control hutch by using a humidity generator. Results show that the developed setup maintains a stable climate during the entire duration of a tomographic scan at different humidities and does not obstruct the sample or hinder the imaging conditions. During the tomographic investigation the sample remains stable in the flow of the air stream and shows typical cell wall swelling and shrinking behaviour depending on the equilibrium moisture content. This new climate cell is now available to all users of the P05 nanotomography instrument for conditioning samples, serving a wide range of scientific applications. Design and implementation of an in situ climate chamber for nanotomography allowing the analysis of moisture sensitive specimens at different relative humidity and temperature settings at the nanotomography imaging station of beamline P05 (operated by Hereon, at the PETRA III storage ring, DESY, Germany).

Digital Volume Correlation (DVC) was used to study the hygroscopic shrinkage in lignocellulosic tissues. For this, small tissue segments of only a few cells were prepared from the endocarp of Hura crepitans fruits, the sclereid cell layer of Pinus jeffreyi pine cone scales, the sclerenchyma fiber sheath of peripheral vascular bundles in Marantochloa leucantha and latewood of Pinus sylvestris . The cells were imaged in a wet and dry state using X-ray nano-holotomography. Subsequently, a DVC analysis was conducted using Avizo™, elastix and MBS-3D-OptFlow, to visualize and quantify their hygroscopic shrinkage and to compare the accuracy of the approaches. The results reveal an anisotropic shrinkage behavior (1) along the cell length compared to radial shrinkage and (2) a greater radial than tangential shrinkage within the cell wall. The accuracy of the DVC results was validated and compared using two artificially deformed datasets (linear and sinusoidal) for controls. A (sub-)voxel accuracy for both controls could be demonstrated for each software with the image registration toolkit elastix performing best. In addition, the abundance of structural features in the cell walls leads to an improved DVC accuracy. Overall, DVC proved to be a viable approach to study the hygroscopic deformation of lignocellulosic tissue samples.

The anisotropic hygroscopic behavior of pine cone scales and its effect on bending motion, with implications for bioinspired actuation, is investigated. Using gravimetric water uptake measurements, synchrotron radiation-based nano-holotomography, and digital volume correlation analysis, inter- and intra-tissue variations of hygroscopic swelling/shrinkage were observed. In addition, the moisture content of pine cone scale tissues was measured as a function of relative humidity. There were distinct differences between tissues and a pronounced hysteresis between sorption and desorption. Finite element analysis was performed on geometries ranging from simplified bilayer models to complex remodeled scales. Simulation results showed an underestimation of the bending of bilayer geometries due to an overestimated contribution of sclerenchyma fiber stiffness. Geometries with discrete fibers embedded in a brown tissue matrix more accurately reproduced the bending angles observed in experiments. This highlights the importance of the chosen material properties and tissue arrangements for predicting pine cone scale bending in silico. By contributing to a deeper understanding of pine cone scale biomechanics, these results also support the development of bioinspired technical applications. Future studies should refine tissue mechanical properties and integrate high-resolution computed tomography-based geometries to further elucidate the mechanisms underlying hygroscopic actuation. This integrative approach will bridge experimental findings with computational modeling and advance plant biomechanics and biomimetic transfer.

The Opuntioideae include iconic cacti whose lateral branch–branch junctions are intriguing objects from a mechanical viewpoint. We have compared Opuntia ficus-indica, which has stable branch connections, with Cylindropuntia bigelovii, whose side branches abscise under slight mechanical stress. To determine the underlying structures and mechanical characteristics of these stable versus shedding cacti junctions, we conducted magnetic resonance imaging, morphometric and anatomical analyses of the branches and tensile tests of individual tissues. The comparison revealed differences in geometry, shape and material properties as follows: (i) a more pronounced tapering of the cross-sectional area towards the junctions supports the abscission of young branches of C. bigelovii. (ii) Older branches of O. ficus-indica form, initially around the branch–branch junctions, collar-shaped periderm tissue. This secondary coverage mechanically stiffens the dermal tissue, giving a threefold increase in strength and a tenfold increase in the elastic modulus compared with the epidermis. (iii) An approximately 200-fold higher elastic modulus of the vascular bundles of O. ficus-indica is a prerequisite for the stable junction of its young branches. Our results provide, for both biological and engineered materials systems, important insights into the geometric characteristics and mechanical properties of branching joints that are either stable or easily detachable.

The development of the branch-stem-attachment of Dracaena marginata was analyzed to clarify how a load-adapted arrangement of mechanically relevant tissues, i.e. the vascular bundles with fiber caps, is established during ontogeny. For this purpose, 3D images of four intact and developing buds of D . marginata were repetitively acquired in vivo within the time span of 180 days using high-resolution magnetic resonance imaging, as this method allows for non-invasive and non-destructive image acquisition. This methodical approach enabled the classification of distinct ontogenetic stages revealing the complex ontogeny of the branch-stem-attachment in D . marginata and the establishment of a load-adapted tissue arrangement within the junction between branch and main stem. This further allows for a first biomimetic abstraction and the transfer into a technical implementation of the form-structure-function principles found in branchings in D . marginata .

Within the Aizoaceae, the genus Delosperma exhibits a vast diversification colonizing various ecological niches in South-Africa and showing evolutionary adaptations to dry habitats that might include rapid self-sealing. Leaves of Delosperma react to external damage by the bending or contraction of the entire leaf until wound edges are brought into contact. A study of leaf morphology and anatomy, biomechanics of entire leaves and individual tissues and self-sealing kinematics after a ring incision under low and high relative humidity (RH) was carried out comparing the closely related species Delosperma cooperi and Delosperma ecklonis, which are indigenous to semi-arid highlands and regions with an oceanic climate, respectively. For both species, the absolute contractions of the examined leaf segments (“apex”, “incision”, “base”) were more pronounced at low RH levels. Independent of the given RH level, the absolute contractions within the incision region of D. cooperi were significantly higher than in all other segments of this species and of D. ecklonis. The more pronounced contraction of D. cooperi leaves was linked mainly to the elastic properties of the central vascular strand, which is approximately twice as flexible as that of D. ecklonis leaves.

In many biomimetic approaches, a deep understanding of the form–structure–function relationships in living and functionally intact organisms, which act as biological role models, is essential. This knowledge is a prerequisite for the identification of parameters that are relevant for the desired technical transfer of working principles. Hence, non-invasive and non-destructive techniques for static (3D) and dynamic (4D) high-resolution plant imaging and analysis on multiple hierarchical levels become increasingly important. In this study we demonstrate that magnetic resonance imaging (MRI) can be used to resolve the plants inner tissue structuring and functioning on the example of four plant concept generators with sizes larger than 5 mm used in current biomimetic research projects: Dragon tree (Dracaena reflexa var. angustifolia), Venus flytrap (Dionaea muscipula), Sugar pine (Pinus lambertiana) and Chinese witch hazel (Hamamelis mollis). Two different MRI sequences were applied for high-resolution 3D imaging of the differing material composition (amount, distribution, and density of various tissues) and condition (hydrated, desiccated, and mechanically stressed) of the four model organisms. Main aim is to better understand their biomechanics, development, and kinematics. The results are used as inspiration for developing novel design and fabrication concepts for bio-inspired technical fiber-reinforced branchings and smart biomimetic actuators.