Photoacoustic imaging (PAI) is a rapidly evolving field in molecular imaging that enables imaging in the depths of ultrasound and with the sensitivity of optical modalities. PAI bases on the photoexcitation of a chromophore, which converts the absorbed light into thermal energy, causing an acoustic pressure wave that can be captured with ultrasound transducers, in generating an image. For in vivo imaging, chromophores strongly absorbing in the near-infrared range (NIR; > 680 nm) are required.

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... tetrapyrroles have a long history in biomedical applications, novel tetrapyrroles and inspired mimics have been pursued as potentially suitable contrast agents for PAI. The goal of this review is to summarize the current state of the art in PAI applications using tetrapyrroles and related macrocycles inspired by it, highlighting those compounds exhibiting strong NIR-absorption. Furthermore, we discuss the current developments of other absorbers for in vivo photoacoustic (PA) applications. Int. J. Mol. Sci. 2020, 21, 3082 2 of 20 causing thermoelastic expansion generating acoustic pressure waves in a megahertz frequency range. When developing exogenous PA absorbers, it needs to be avoided that the excited states relaxation by light emission (fluorescence) or catalytic production of singlet oxygen from triplet oxygen, thereby causing a loss to the PA generation and potentially toxic effects on the tissue. Commercial PAI devices enable simultaneous US imaging (B-mode), allowing co-registration of anatomical information, to visualize tissue structures and to locate pathological sites. Three dimensional (3D) PA tomography (PAT) is a commonly used detection, in which several planes of 2D-images are fused into a 3D-image, which still allow the separation of PA signals into respective absorbers; e.g. multispectral optoacoustic tomography (MSOT), where multiple excitation lasers are used in a time-shared fashion to excite the contrast agents and the endogenous background, and spectrally differentiate those signals [9] . With advances in computing power, the PAI devices provide the molecular and functional information on the fly and enable real-time detection [10] . PAI methods are used for in vivo preclinical imaging of a variety of pathological conditions, predominantly in the cancer research, [11] [12] [13] [14] and several clinical translational studies are ongoing [10, [15] [16] [17] . Some indications like, sentinel lymph nodes, [18, 19] arthritis, [20] burn wound healing, [21] Duchenne muscular dystrophy, [22] inflammatory-bowl disease, [23] metabolism of brown fat, [24] periodontal pockets, [25] and several other ongoing investigations [26-30] have proven the added value of PAI to the clinical routine. PA Devices and Noninvasive Imaging Procedures Currently, several commercial PA devices are available from different suppliers (Figure 1 ). These devices are equipped with a light source and ultrasound transducer, using interactive software for fast image analysis. Depending on the optical illumination and acoustic detection configurations, imaging can be performed in 2-D, 3-D tomography, 2-D high resolution microscopy settings and as mesoscopy. The commercial devices, useful for in vivo imaging, are equipped with a nanosecond pulse (Nd:YAG) laser with a frequency of 10 to 20 Hz, which provide pulses with a duration between 7 to 10 ns, and a peak pulse energy of 18 to 26 mJ/cm 2 in the wavelength range of 680-1100 nm. This energy is within the maximum permissible exposure (MPE) range, which is dependent on the wavelength, pulse frequency and exposure time, e.g. between wavelength of 700 to 1050 nm with a pulse duration of 1 to 100 nanoseconds MPE is 20·10 (2(λ−700)/1000) mJ/cm 2 , as defined by the American National Standards Institute (ANSI) [31] .