# Optics Based Label-Free Techniques and Applications in Brain Monitoring
## 4 Current PAI Technology Used in Brain Imaging
#### Brain Studies Utilising PAI  
An early-stage PAT system in structural and functional imaging mice brain vasculature was reported by [PERSON] et al. [130]. Since then, many kinds of PA imaging systems were developed and many a great number of articles about brain imaging and monitoring are published. Recently, [PERSON] et al. [30] employed PACT with a 512-element ring transducer array to perform functional analysis of resting-state functional connectivity (RSFC) on mouse. RSFC is of great importance in the study of stroke, AD, multiple sclerosis, autism, epilepsy, etc. Compared with other functional imaging techniques, functional PAT (fcPAT) provides higher resolution, and the ability of molecular imaging when combining with biomarkers. [PERSON] et al. [31; 131] described a more real-time miniature cap-like wearable PAT system, which can non-invasively image blood vessels at a depth of 5 mm. The system can be used for behavior, cognition and preclinical brain disease studies using animal models. [PERSON] et al. [32] developed a PACT system for non-invasive and label-free mapping of the microvascular network of the mouse brain and to study its hemodynamic activities. Spontaneous neural activities in the deep brain were studied by monitoring the concentration of Hb in blood vessels and strong inter-hemispherical correlations between several functional regions were observed during an epileptic seizure. [PERSON] et al. [132] developed a volumetric multispectral PAT platform for imaging neural activation deep in scattering brains. Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing a genetically encoded calcium indicator. The study showed that PA has the ability to directly track neural dynamics while overcoming the longstanding penetration barrier of optical imaging in scattering brains. PAI has a wider range of application in deep monitoring, diagnostic and therapy of brain diseases and activities by sensing injected exogenous agents such as nanoparticles and dyes. [PERSON] et al. [133] designed and synthesized biocompatible conjugated polymer nanoparticles (CP_NPs) for highly efficient PAI of orthotopic brain tumors in the second NIR window. The CP_NPs had excellent photostability, high imaging contrast and penetration at 1064 nm. Moreover, these CP_NPs can be used in dual NIR-II fluorescence and photoacoustic imaging, which provides the possibility to noninvasively map deep microscopic brain tumors after focused ultrasound (FUS) induced BBB opening [134]. Usually, PAM has 3-mm imaging depth. To improve its imaging depth, mesoionic dye A1094 encapsulated in Arg-Gly-Asp-modified hepatitis B virus core protein (RGD-HBc) was designed and synthesized for effectively improving PAM imaging depth [135]. Nine-fold PA signal amplification in vivo was reported, and imaging depth is up to 5.9 mm with acoustic resolution. This gives a promise of A1094@RGD-HBc for diagnostic imaging and precise delineation of brain gliomas in clinical applications. Recently, PAT is frequently used with exogenous agents in research related to BBB. Together with bone mesenchymal stem cells (BMSCs) to be labeled with modified Prussian blue, excellent near-infrared dyes and photoacoustic contrasts, PAT can improve observation and efficient therapy of traumatic brain injury (TBI) and rehabilitation [136]. The BMSCs proved to be capable of overcoming BBB with enhanced delivery of Prussian blue particles to the brain parenchyma. These labeled BMSCs were clearly observed by PAT to home to the damage region and repair the ruptured vasculature. Moreover, the wound treated by the BMSCs exhibited a much faster recovery speed than that without treatment. These potentially provide a noninvasive and high-resolution approach to image TBI, monitor recovery process. Another recent study by [PERSON] et al. [137] used NIR voltage-sensitive dye (VSD) together with functional PAI for the transcranial recording of neural activity in the rodent brain.