*3.1. Computed Tomography (CT)*

In the clinical field, CT imaging has extensive applications in inflammation, angiography, cancer detection, and the evaluation of bone regeneration or toxicities. It provides useful anatomical details, but exposure to high radiation often limits the number of scans performed in the same patient. In preclinical research, this methodology, besides being particularly useful for the assessment of skeletal and lung abnormalities and cardiac function, is considered a robust technique for the quantitative evaluation of angiogenesis associated with solid tumors. The small animal CT has a high spatial resolution (up to 10 μm, in vivo) and relatively short imaging times. However, the employment of clinically-used contrast agents limits the repeated imaging in the same animal, in particular, due to the iodinated contrast, which is rapidly cleared from the blood, and therefore, should be administered repeatedly. Moreover, high dosages of this agent may result in nephrotoxicity, and its use may produce an ionization effect, resulting in radiation damage through reactive oxygen species [8,17,81–84].

## *3.2. Positron Emission Tomography (PET)*

The PET has become a powerful tool for clinical diagnosis and preclinical research, and it plays an essential role in oncological research in monitoring metabolism, gene expression, cell proliferation, angiogenesis, hypoxia, and apoptosis, as well as for drug development [85]. Indeed, this modality, in the clinical setting, is used for tumor staging, to evaluate tumor response to therapies through the use of an radiolabeled imaging agent, such as [18F]-2-fluoro-2-deoxy-glucose [18F]-FDG) for glucose metabolism or [18F]-fluoro-3'-deoxy-3'-L-fluorothymidine [18F]-FLT) for cell proliferation [86]. The PET also has a wide range of applications in preclinical research; for example, it allows investigating physiological

and molecular mechanisms of human diseases, and evaluating novel radiolabeled imaging agents, and the biodistribution and the efficacy of new drugs in suitable animal models [87]. As for clinical scanning, preclinical PET devices provide excellent sensitivity (from 2% to 7%, depending on the energy window) and spatial resolution (1.2 mm, on average), but the main disadvantages are the lack of an anatomical parameter, usually overcome by associating a CT scan or MR scans. Moreover, the short half-life of most radioisotopes requires strict coordination between radiotracer production, delivery, and use in small animal models [83,84].

#### *3.3. Single-Photon Emission Computed Tomography (SPECT)*

Single-photon emission computed tomography (SPECT) is a functional, nuclear medicine technique based on the detection of gamma photons emitted by a radionuclide during its decay. SPECT employs a gamma camera, composed of detector crystals and a lead or tungsten collimator with multiple elongated holes—pinholes, which rotates around the subject and acquires multiple cross-sectional images. This technique is similar to PET, and it is widely employed in clinical routines, but it is less sensitive than PET. Indeed, the spatial resolution of clinical SPECT (8–10 mm) is lower than that of clinical PET (4–6 mm) [17,83]. The main advantage is the availability of several radiotracers ( 99mTc, 67Ga, 111In, and 123I), which have relatively long half-lives compared to most PET radiotracers. In oncological research, SPECT tracers targeting angiogenesis, hypoxia, acidosis, and metabolic activity have been developed and applied [82]. In preclinical investigations, micro-SPECT has a higher spatial resolution (0.35–0.7 mm), compared to micro-PET, and as mentioned, the longer half-life of SPECT tracers is advantageous in terms of production and transport. On the other hand, in longitudinal studies, researchers have to wait for complete decay accordingly. The SPECT's main application is the study of the biodistribution and kinetics of novel radiopharmaceuticals. Indeed, preclinical PET scanners and radiopharmaceuticals showed a stronger ability to study tracers' kinetics compared to SPECT counterparts [82,83].
