Micro-computed tomography (µ-CT) is an X-ray-based scanning imaging tool that generates 2D trans-axial projections of a specimen [108,109]. While µ-CT is classified in this work as a photon-based technique, X-rays derive from the electron interactions within a high-energy electromagnetic beam [110]. In the µ-CT equipment, the sample is placed on a rotational stage and exposed to an X-ray source, and the passing light is captured by an X-ray detector. The passing X-rays can be attenuated (i.e., absorbed or scattered) by the sample thickness, density, and composition, providing phase contrast to the structures and components [111]. The µ-CT images can be reconstructed in 3D with an up to 1 μm voxel (3D pixel) resolution, making this a high-resolution technique. As an X-ray-based imaging method, µ-CT has historically been reported as a tool for reconstructing bone microarchitecture [112,113,114,115]. Studies on cell-loaded and cell-free hydrogels have used this technique to detect hydrogel mineralization both in vitro and in vivo [116,117,118,119]. The µ-CT data can be used to determine the pore size and fiber thickness in order to reconstruct the polymer network in 3D, demonstrating the pore interconnectivity [102,108]. Hydrogel degradation tests employing µ-CT in vitro demonstrate an increase in the pore sizes and porosity percentages [105,120,121,122]. While µ-CT is regarded as both non-destructive and non-invasive, exposure to high current and voltage levels will invariably dehydrate the sample [123]. High voltage levels are common when generating high-resolution images, but they are costly and result in a prolonged imaging time. To prevent structural damage during imaging, some reports recommend specimen fixation or the use of (freeze-)dried samples [116,121]. Thus, it is not uncommon to validate hydrogel µ-CT data using SEM data derived from dry specimens [105,123,124,125]. There are other limitations of µ-CT, as the hydrophilic nature of hydrogels provides a low-phase separation contrast (Figure 4) [119]. Radiopaque agents can be coupled with the polymer backbone or solubilized and left to soak with the hydrogel to improve said contrast [119]. Contrast agents allow us to discern between the hydrogel fibers and porosity and have been used to investigate hydrogel degradation in vivo and in vitro [126,127]. Reportedly, osmium tetroxide and uranyl acetate, or a combination of uranyl acetate and lead citrate, enabled a good contrast resolution and 3D reconstruction of collagen-I hydrogels [128]. The use of metal nanoparticles (e.g., Au) has also been reported to improve the hydrogel contrast [119] or the visualization of the spatial distribution of the cells within. Other limitations include vibrations in the µ-CT’s rotatory stage, which can cause motion artefacts by displacing the sample if it is not properly fixed—a common challenge with wet materials [123]. Information on the in-depth specifications and further limitations of µ-CT in materials research can be found elsewhere [111,123].

Confocal laser scanning microscopy (CLSM) is a photon-based fluorescent imaging technique. For visualization, CLSM requires that proteins and structures are stained or coupled with fluorophores: organic molecules that can emit light (λ_em) upon light excitation (λ_ex) [129]. The CLSM optical resolution is determined by the wavelength of the laser. This wavelength is chosen according to the absorption spectra of the fluorophores, and multiple lasers can be used at the same time or in succession to capture multi-color fluorescent images [130]. Unlike conventional fluorescent microscopes that illuminate the entire specimen, CLSM uses point illumination, typically a laser beam, and a pinhole in front of the detector to eliminate most of the background blur, greatly increasing the optical resolution [131]. As only a single point of the specimen is illuminated, the samples must be scanned over a specified region to produce 2D or 3D fluorescent images.

CLSM is an invaluable tool used to visualize the structures of living cells within hydrogels [132]. For the assessment of the hydrogel microarchitecture, these can be fluorescently dyed [58], although certain polymers are naturally auto-fluorescent (e.g., chitosan), allowing dye-free imaging [133]. CLSM yields high-resolution 2D and 3D images (Figure 4), revealing the polymer network microarchitecture in great detail, crucial in the determination of the pore size and porosity of hydrogels in their swollen state (Figure 5) [57,58,59]. Overall, CLSM agrees with SEM in regards to the pore size decreasing with increasing polymer concentrations [58]. CLSM images can be segmented into a polymer phase and a fluid phase [42] (Figure 4, orange). During analysis, spheres can be fitted into the fluid phase (Figure 4, blue), and their diameter is measured to determine the pore diameter. The pore diameter, together with the median values of all the detected pore diameters, is defined as the pore size of a particular sample [42]. Applying the same principle to the polymer phase can determine the average thickness of individual fibers (Figure 5A). Additionally, CLSM images can be used to quantify local deviations in the microarchitecture due to nodes formed during the polymerization of collagen-I hydrogels. Moreover, data from fluorescently labelled collagen-I hydrogels showed that fiber diameter and fiber length are heavily influenced by pH, irrespective of the hydrogel concentration. In contrast, the pore size remained unaffected by pH [58].

Data from CLSM applied to cell-loaded hydrogels can be used to detect local cell-induced microenvironmental changes during migration through an inhomogeneity parameter [57]. This inhomogeneity parameter can explain differences in cell migration that cannot be explained using the pore size and mechanical properties alone. Therefore, elucidating these differences has led to significant insights into the role and adaptation of the microenvironment during cell migration [57].

CLSM has some limitations, the most important being the resolution limit due to optical diffraction limits. CLSM employs lasers with excitation wavelengths of several hundred nanometers, and the excited fluorophores commonly emit light at a higher wavelength. For example, collagen fibers in polymerized matrices possess a wide range of diameters, ranging from below 100 nm [134] to nearly 1 µm [58]. Using a λ_ex 561 nm-laser and recording λ_em 580 nm, objects as small as 290 nm can be distinguished. Thus, this technique [42] must be considered an overestimation, as the fiber thickness can be below the optical diffraction limit. However, due to multiple post-processing steps, the analysis can be considered as quasi-pixelwise. Another important consideration is the efficacy of the fluorescent dyes used. Poor staining might result in a low signal-to-noise ratio or even the inhomogeneous staining of a hydrogel, resulting in limitations in the dynamic range. Thicker and, thus, brighter fibers may overlay smaller fibrils, or vice versa, with larger fibers losing detail when overexposed. Lastly, the choice of a spherical space estimator might not be suitable for a specific problem. For a robust estimate of the cell migration, spheres can be considered a suitable shape. However, other problems can make it necessary to choose other shapes, such as cubes. Fitting shapes into the hydrogel segmentation might be entirely superfluous, and polymer content calculations based on the precise segmentation may provide a better parameter.