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International Journal of Bioprinting PAI for 3D bioprinted constructs

laser excitation. 36–38 PAI resolves optical contrasts from Additionally, we address practical considerations that
the emitted acoustic signal, 39–42 which experiences less should be factored in for more complete integration of PAI
diffraction compared to optical rays in the tissue. 43–46 In into the fields of 3D bioprinting, with our anticipation of
addition to the imaging exogenous contrast of a printed an increasingly exclusive role for PAI as bioprinting evolves
artificial construct, PAI is also capable of delineating towards more complex hierarchies.
endogenous chromophore distribution within the tissue,
such as melanin, hemoglobin, fat, and collagen, unlike 2. Unveiling physiological landscapes of
conventional optical imaging methods that require tissue via PAI
destructive processes such as tissue slicing, fixation,
staining, and fluorescent labeling. 2.1. Flexible tissue contrast of PAI with a variable
light source
One important feature of PAI is that its spatial The core objective of bioprinting is to fabricate
resolution and imaging depth can be scaled by varying microarchitectures that can initiate cell growth and
the hardware configuration, ranging from sub- facilitate sufficient maturation to drive clusters of cells
micrometer to millimeter scales. The spatial resolution towards functional tissue differentiation. Within this
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of PAI is determined by the size of either the optical context, bioimaging plays a crucial role in monitoring
excitation beam or the receiving acoustic beam. This the physiological changes occurring within bioprinted
principle primarily classifies PAI techniques into optical constructs, encompassing vital processes such as cell
resolution photoacoustic imaging (OR-PAI) and acoustic proliferation, angiogenesis, and intra- or inter-cellular
resolution photoacoustic imaging (AR-PAI). Due to metabolic dynamics. Cell proliferation serves as a crucial
the proportionality between the diffraction limit and indicator of the viability of printed biomaterials, both in
the wavelength, optical beams (beam size <1 μm) can vitro and in vivo, and is determined from changes in the
be more tightly focused than acoustic beams (beam cell population. The observation of angiogenesis enables
size >50 μm). 47–50 Conversely, acoustic waves suffer less the assessment of biocompatibility after the in vivo
attenuation with less absorption or scattering than optical implantation of printed biomaterials. It can also indicate the
rays, allowing AR-PAI (1–80 mm) to achieve greater formation of microvascular resulting from enhanced tissue
imaging depths than OR-PAI (<1 mm). 51–60 Given the regeneration, particularly in functional 3D bioprinting
need to strike a compromise between imaging resolution applications. Finally, bioimaging facilitates the observation
and depth, the PAI technique should be chosen based on of functional maturation and differentiation into complete
its key utility in 3D bioprinting. For example, OR-PAI is tissues, enabling the visualization of metabolic changes at
essential for microscopic cellular-level examinations, such the organelle or tissue level.
as tracking cellular growth or functional maturation in Given the uniqueness of the optical absorption
vitro. In contrast, AR-PAI is more suitable for mesoscopic spectrum of chromophores, PAI excels in specifying
or macroscopic investigations at the tissue or organ level, cellular organelles or tissue constituents of interest by
such as longitudinal monitoring of implanted or engrafted modulating the wavelength of the light source (Figure 2).
3D constructs in vivo. Overall, the ability to discover Ultraviolet-photoacoustic imaging (UV-PAI) allows for
voluminous and complex biological structures without the visualization of cell nuclei populations via the high
destruction is invaluable in the field of 3D bioprinting, absorption of nucleic acids, such as ribonucleic acid (RNA)
where precise evaluation of tissue architecture and and deoxyribonucleic acid (DNA) under UV irradiation,
functionality is paramount.
facilitating the identification of cell proliferation. Second,
In this comprehensive review, we introduce PAI as blood vessels can be imaged using visible or near-infrared-
a novel optical imaging technique that provides optical photoacoustic imaging (VIS/NIR–PAI), leveraging the
contrast to an unprecedented depth, capable of visualizing strong absorption properties of hemoglobin within red
both the morphological and physiological features of blood cells (RBCs). VIS light source, typically a 532 nm
3D-bioprinted constructs. The versatility of PAI across green diode laser, is predominantly used for microscopic
different spectral ranges is demonstrated, presenting imaging of superficial structures. On the other hand, NIR
various spectral contrasts from tissues spanning the light, often referred to as the “optical window,” encounters
ultraviolet (UV), visible (VIS), near-infrared (NIR), and less absorption and scattering by biological tissues, allowing
mid-infrared (MIR) regions. We further summarize for deeper tissue imaging. Finally, proteins and lipids in
the collaborative bioprinting research to date, which the protoplasm are strongly absorbed in the MIR range
functionally implements PAI for obtaining the architectural (greater than 1500 nm), as detectable by mid-infrared
information of bioprinted constructs and their noninvasive photoacoustic imaging (MIR-PAI). In this section, we
longitudinal monitoring post-implantation in vivo. introduce PAI techniques categorized by their wavelengths,

Volume 10 Issue 4 (2024) 3 doi: 10.36922/ijb.3448