*2.8. Optical Coherence Tomography*

In addition to fluorescence and bioluminescence, which only provide 2D image information, recent advances in optical imaging technologies have allowed for the 3D visualization of tissue structure through the measurement of the interference and coherence between signals reflected from the object and reference signals, known as optical coherence tomography (OCT) [11]. Due to this unique property, OCT can provide anatomical information of the object with sub-millimeter penetration depth [10]. OCT can be used with a variety of light sources, ranging from NIR to visible light [125,126]. In the field of TE, OCT has been used to investigate the geometrical parameters of 3D sca ffold architecture, including porosity, surface area, pore sizes, and pore interconnectivity [127,128], as well as remodeling and degradation of polymer structures for specific applications such as vascular grafts [129]. OCT can also be used to asses cell viability, proliferation, distribution, morphology, and function within a cell-laden hydrogel and sca ffold (Figure 9) [130]. Advancement in phase-based OCT has also been able to provide contrast between cells and the surrounding hydrogels, thus allowing to achieve a greater understanding of the cell–ECM interactions [128]. Furthermore, a combination of OCT with Doppler velocimetry has been used to characterize flow in engineered tissues, such as artificial blood vessels, by increasing the obtained contrast compared with conventional OCT [11]. Thereupon, OCT has been widely used in conjunction with automated 3D fabrication techniques, such as 3D bioprinting [131], as a high-resolution, noninvasive, label-free method, enabling cellular imaging at di fferent levels for TE applications (Figure 10).

**Figure 10.** (**<sup>a</sup>**–**i**) OCT design and the tracking of cells in a 3D bioprinted sca ffold seeded with cells. Reproduced with permission from Ref. [127].

### *2.9. Photoacoustic Imaging (PAI)*

Photoacoustic imaging (PAI) leverages the photoacoustic e ffect produced by pulsed non-ionizing lasers in tissue to reconstruct an image. For this method, the pulsed laser energy causes heat-induced, elastic tissue expansion, of which emits ultrasonic waves in the MHz range. Via ultrasound transducers, these waves are detected and electronically processed to output a final picture (Figure 11) [132]. PAI diverges based on the acquisition method into photoacoustic microscopy (PAM—focused scanning) and photoacoustic tomography (PACT—inverse reconstruction) [133]. Overall biomedical applications of this technique vary from visualizing macroscopic structures (e.g., small animals to tissues) to microscopic structures (e.g., cells to organelles), with associated contrast agents [133,134]. The advantages of PAI include its non-invasiveness, non-destructiveness, macro to microscale versatility, and compatibility with established imaging modalities. Conversely, PAI challenges include merging optical and acoustic signals, limited scanning speed for wide fields of motion and optimized mathematical models across measurement scales [132,133].

**Figure 11.** Overview of the physics and processing involved in photoacoustic imaging (PAI) (**left**), and a sample of 2D and 3D vasculatures acquired via PAI from hemoglobin and melanin emissions without contrast agents (**right**, **<sup>a</sup>**–**<sup>c</sup>**). Reproduced with permission from Ref. [132].

Applications of PAI in TE and 3D (bio)printing are beginning to expand. For instance, Cai et al., compared the resolution of microcomputed tomography to PAM technique in sca ffolds of poly(lactic-co-glycolic acid) incorporated with single-walled carbon nanotubes. They demonstrated commensurate porosity measurements under physiological conditions [134]. In another study, acoustic and physiomechanical properties of 3D bioprinted poly-(ethylene glycol)-diacrylate sca ffolds were quantified using an ultrasound pulse echo technique [135]. Hu and Wang have shown micrometer-level resolutions of microvasculatures capable of both capturing geometric and hemodynamic information, such as blood oxygenation [136]. This can be extremely useful in monitoring the oxygenation of implanted TE constructs. Much of PAI research is currently focused on optimizing contrast agents for high fidelity imaging for eventual in vivo applications [137–139].

Contrast agents are not always needed due to the PA emissions of already present hemoglobin and melanin. However, to penetrate beyond 1 mm depth, contrast agents that absorb NIR waves are optimal [140]. Metallic (e.g., gold and copper selenide-gold [141]), organic (e.g., carbon tubes and graphene oxide [138]), and semiconductor (e.g., semiconductor polymers and quantum dots [137]) nanoparticles of varying orientations have been employed. Of the three, organic particles exhibit size-independent properties and improved biocompatibility and biodistribution, especially with surface neutralization via encapsulation. This enables multiplexed imaging with customized organic nanoparticles [140]. Overall, PAI development holds much promise in monitoring the TE systems.
