## 3 Exogenous Reagents for PAI  
Similar to other techniques, in deep tissue absent of endogenous absorbers or where the absorbing coefficients are low, the use of PAI can increase imaging contrast in deep tissue without endogenous absorbers or a sufficiently strong absorbing coefficient. This occurs through the introduction of exogenous absorption materials, such as small molecule dyes, metallic nanoparticles, single-walled carbon nanotubes, semiconducting polymer nanoparticles and quantum dots in the interested location. If the exogenous contrast agents can be bio-conjugated and attached with specific cells or tissues, they can also enable targeted molecular imaging, in addition to imaging contrast enhancement. Hence, these exogenous agents may greatly extend the preclinical and clinical application of PAI and other resembling techniques. Details of the exogenous contrast agents for PAI enhancement can be found in recent review papers [24; 60], here we focus on the progress made in brain imaging using contrast agents over the past five years. Deep imaging of brain and body tissues using PAI requires NIR wavelengths due to the minimal optical attenuation caused by low tissue absorption/scattering. However, low tissue absorption results in a weak PA generation. Moreover, the intrinsic biomarkers of many diseases cannot generate PA signals in the NIR range. This means it becomes necessary to develop exogenous PA contrast agents to improve the SNR of PAI in deep tissues and/or the accurate detection of diseases in living systems. Conjugated polymer nanoparticles (CPNs) are increasingly attracting interest as PAI contrast agents, due to their inherent advantages, which include a large absorption coefficient (compared with carbon nanotubes), excellent photostability (better than gold nanorods) and good biocompatibility. Recently, [PERSON] et al. [61] rationally designed three donor-acceptor-structured conjugated polymers and prepared the corresponding CPNs. The chemical structures of these CPNs were nearly identical, except for the heteroatom of the acceptor moiety, which was systematically varied from oxygen to sulphur to selenium. The results indicate that the absorption coefficient, and in turn the PA signal intensity, decreased when the heteroatom changed from oxygen to sulphur to selenium. As these CPNs exhibit a weak fluorescence and a similar photothermal conversion efficiency (\(\approx\)70%), their PA intensities were approximately proportional to their absorption coefficients, which was demonstrated by in vivo brain vasculature imaging. The experiment also evidenced that CNPs offer good photostability and do not have toxicity. Research has suggested that molybdenum disulphide (MoS2) nanosheets have a good physiological stability and biocompatibility, higher optical absorption than gold nano-rods and graphene derivatives in the NIR wavelength, and excellent enhanced permeability and retention (EPR) effects for tumour targeting. As a result, [PERSON] et al. investigated MoS2  
Figure 4: Performance evaluation of trained U-Net kernel based on in vivo sheep brain images. (**A**), US image along the sagittal plane. (**B**), PA image generated with a low energy laser; (**C**), deep learning enhanced PA image in (**B**). S, skin; LV, lateral ventricle; TV, third ventricle; TH, thalamus; VA, vasculature. Yellow contours indicate some of the structures that were not present in B (Reprinted with permission from Ref. [59]. 2020, Wiley-VCH). nanosheets using differently layered nanostructures and observed that single-layered MoS2 presents a better PAI sensitivity than its multiple layered counterparts, due to its higher NIR absorption and superior elastic properties [62]. An intravenous administration of S-MoS2 to both U87 subcutaneous and orthotopic tumour-bearing mice revealed that a tumour located 1.5 mm below the skull can be clearly rendered by PAM in vivo.