Respiratory viruses possess strong infectivity, rapid transmission, short incubation periods, and sudden onset of illness. These features have led to widespread global transmission, significantly affecting the health of children worldwide. In addition, these viruses have caused significant economic losses and casualties in various countries. Antibiotics are commonly used to control respiratory infections in humans. Therefore, accurate and rapid understanding of the course of respiratory infections is the foundation for selecting a treatment plan.

In biomedical imaging, various imaging methods can reveal microscopic and macroscopic phenomena within organisms. These methods include magnetic resonance imaging (MRI), computed tomography, positron emission tomography, ultrasound (US) imaging, optical coherence tomography, and fluorescence imaging. These technologies provide rich information, thereby contributing to a comprehensive understanding of the characteristics of respiratory infections and supporting the development of rational treatment plans. Owing to limitations in specificity, resolution, and radiation, these imaging techniques lack the ability to accurately image biological structures in the early stages of disease development. In this study, the noninvasive, deep-penetrating, and high spatial resolution advantages of photoacoustic (PA) imaging (PAI) are utilized. This is combined with the excellent fluorescence properties of the exogenous contrast agent indocyanine green nanoparticles (nano-ICG) in the near-infrared region and the high expression of macrophages during inflammation. This combination enables the visualization of the development of respiratory inflammation.

Through the establishment of animal models and in vivo experiments, we quantitatively evaluate the macrophage expression in acute respiratory infections, as shown in Fig.1. Research on PAI is expected to provide a new approach for the noninvasive quantitative assessment of inflammation in acute respiratory infections.

This study uses a respiratory inflammation mouse model for photoacoustic imaging. Initially, the mice are anesthetized using isoflurane with volume fraction of 1.5%, followed by the instillation of lipopolysaccharide (LPS) solution into the mouse respiratory tract to construct the respiratory inflammation model group after two days. Five mice are selected from the Control and Model groups for further studies. Subsequently, the ultraviolet absorption spectra and cytotoxicity of nano-ICG materials are studied under irradiation at different wavelengths. The internalization dynamics of macrophages after nano-ICG injection are investigated. Finally, a PA-US dual-mode small animal imaging system is used to image different groups (Control and Model groups). Imaging is conducted before nano-ICG instillation and when the post-injection time is 15, 30, and 60 min in each group of mice. PA and US data collected from the experiment are subjected to offline quantitative analysis using Vevo Lab Software 3.2.0 to observe the overall respiratory inflammation under PAI.

Transmission electron microscopy is used to characterize the shape and size of the exogenous contrast agent, nano-ICG. As shown in Fig.2 (a), Nano-ICG has an average size of approximately 65 nm with a round shape and aggregated distribution. Subsequently, the cell counting kit is employed to evaluate the in vitro viability of macrophages, and the absorbance of each well is determined using enzyme-linked immunosorbent assay, as shown in Figs.2(b) and 2(b). The internalization of nano-ICG at different time points after injection is observed using confocal fluorescence microscope, as shown in Fig.3. These results indicate that nano-ICG continue to be internalized by the macrophages within one hour after injection. Additionally, laser confocal microscope images exhibit a positive correlation between the uptake of nano-ICG by macrophages and time. After engulfing the nanoparticles, the imaging effect of macrophages becomes more prominent. Within the first 15 min after nano-ICG injection in mice, Model group exhibits an enhanced trend in the PA signal compared with the normal group. In Control group, the PA signal of nano-ICG exhibits a decreasing trend over time, whereas in Model group, the corresponding PA signal continues to increase. After 30 min, the PAI images of Control and Model groups exhibit more noticeable contrast. After 60 min, Model group exhibits the strongest PA signal, showing a more significant contrast than Control group, as shown in Fig.4(a). In Control group, the amount of nano-ICG in the mouse airways continuously decreases with increasing post-injection time, as shown in Fig.4(b). In Model group, the quantity of nano-ICG on the mouse airway wall increases continuously with the post-injection time, as shown in Fig.4(c). These results indicate that nano-ICG can effectively reflect the degree of development of inflammatory cells on the respiratory wall when the post-injection time is 60 min. Three-dimensional PAI images of respiratory inflammation provide more accurate information on respiratory wall inflammation, as shown in Fig.5(a). The coronal images generated by two-dimensional PAI scans, indicate the presence of inflammatory cell aggregation in the respiratory tract at that position. Figure 5(b) validates the accuracy of three-dimensional PAI images by showing images of inflammatory and non-inflammatory cells in the respiratory tract using an in vivo imaging system (IVIS) for small animals. Although PAI can visually present respiratory inflammation, some mice must be euthanized for pathological sectioning and staining to gain a more comprehensive understanding of the morphological and structural changes in inflammation. Histological results are shown in Fig.6. Control group sections exhibit a light pink color in the airways with no thickening on the inner side of the tube wall and smooth and regular surfaces without apparent lesions. In contrast, Model group sections exhibit noticeable bleeding, significant swelling, scattered bleeding points on the surface, infiltration of inflammatory cells on the inner side of the tube wall, and increased secretion into the lumen, consistent with the imaging structures of PAI.

This study successfully establishes a mouse model for acute respiratory inflammation and utilizes nano-ICG to observe respiratory inflammation, confirming the feasibility of evaluating inflammation using PAI. The PAI results for inflammation in the model are consistent with the pathological and IVIS results. This research provides new methods and insights for assessing respiratory inflammation. In summary, PAI is widely applicable to respiratory inflammation research because of its unique imaging capabilities, non-invasiveness, and high resolution. This study provides strong support for a deeper understanding of the development of respiratory inflammation and evaluation of treatment effectiveness.