3. Current Insights
Small-animal imaging has become a fundamental technique for the development of new diagnostic or therapeutical radiopharmaceuticals. Indeed, currently, pre-clinical imaging of animal models represents an invaluable tool in studying the etiopathogenesis of and therapeutic responses in various human pathologies such as neurological, cardiovascular and oncological diseases 
. Molecular imaging techniques can be used to assess biological processes at the cellular and molecular levels, enabling the detection of disease in very early or pre-symptomatic stages, and to estimate the efficacy of novel therapies in individual patients 
. The assessment of biological properties of tumours, such as metabolism, proliferation, hypoxia, angiogenesis, apoptosis, and gene and receptor expression, contributes to the realization of precision medicine 
, owing to the possibility of monitoring physio-pathological processes in vivo, detecting therapeutic responses, identifying non-responders at an early stage, and enabling the switch to novel therapeutic approaches 
. In this context, PC represents a unique model for the realization of new protocols of personalized medicine. Indeed, PC is a very heterogeneous disease, and contemporary management is focused on identification and treatment of the prognostically adverse high-risk tumours while minimizing overtreatment of indolent, low-risk ones 
. In recent years, imaging has gained increasing importance in the detection, staging, posttreatment assessment and detection of recurrence of PC 
. Several imaging modalities, including conventional and functional methods, are used in different clinical scenarios with their very own advantages and limitations. Thus, several groups are involved in the development of new radiopharmaceuticals for both the diagnosis and therapy of PC. To these aims, some laboratories now have a combination of different small-animal imaging systems, which are being used by biologists, pharmacists, physicians and physicists.
Unfortunately, the number of laboratories equipped with innovative small-animal imaging systems are currently very few, due to the high costs of these scientific devices. This fact often precludes the development of several promising radiopharmaceuticals. Thus, the enhancement of an instrumental armamentarium available for researchers could significantly increase the chance of success of pre-clinical investigations based on the identification of new radiolabelled molecules.
For several years, the in situ detection of radiolabeled molecules has been performed by using film or film emulsion (conventional autoradiographic analysis). Despite the fact that the spatial resolution obtained with these devices is very good, the sensitivity of film for low activity levels is poor, due to the low x-ray/β particle detection efficiency. According to this, film autoradiographs frequently must take several days to produce a satisfactory image. In addition, the limited dynamic range of film can cause under- or over-exposure of parts of the image. Therefore, better autoradiography systems based on digital position-sensitive detectors have been developed. Among these, the most sensitive are phosphor imaging plates 
, multiwire proportional chambers 
, scintillating optical fibres 
, microchannel plates 
, silicon strip detectors 
, and silicon or gallium arsenide pixel detectors 
. Moreover, in the last years, extremely sensitive digital autoradiographs have been developed both for quality control and in vivo research.
Starting from these considerations, in this study, the potential of a digital autoradiography system equipped with an SR screen has been evaluated to characterize 18F-PSMA inhibitor biodistribution in a PC mouse model within xenograft tumours and mouse organs. In addition, a multidisciplinary investigation including histopathological analysis was performed to study radiopharmaceutical behavior at the sub-cellular level.
A digital autoradiography system is a very versatile and sensitive device for radioisotope imaging, replacing film autoradiography 
. This system has been designed for a great variety of applications, such as the analysis of purity for radiopharmaceuticals, nucleotide metabolism studies, in vitro imaging of tissue sections and also gene and protein expression studies 
. In fact, it can image and quantify activity distribution of different radionuclides (photon-, β- and α-particles emitting).
An SR phosphor screen is a flexible support film formulated with the finest grade of barium fluorobromide and containing traces of bivalent europium (BaFBr/Eu2+) phosphor crystals, which acts as a bioluminescence center to provide the best resolution. When the screen is exposed to a radioactive sample, the energy of the radioisotope ionizes the Eu+ 3 to Eu2+, liberating electrons which are trapped in the bromine vacancies 
. Subsequently, the exposed SR screen, wrapped around the carousel of the photometer reading device, is scanned by a focused red light laser beam (633 nm); the laser-stimulated luminescence releases blue light photons (390 nm) which are detected by a photo-multiplier tube (PMT) and converted to electrical signals expressed as DLU. The SR screen was scanned in a few minutes to create a high-resolution digitized image of the locations and intensity of the radioactivity in the sample, which is quantified by OptiQuantTM image analysis software and stored for future reference.
The data here reported showed that the digital autoradiography system is suitable to analyse the biodistribution of an 18F-PSMA inhibitor in both whole small-animal bodies (mice) and in single organs. Specifically, the exposure of both whole mouse bodies and organs on the SR screen surface allowed the radioactivity of the PSMA inhibitor distributed in the tissues to be detected and quantified. It is noteworthy that data obtained by using the digital autoradiography system were in line with the value of measurement detected by the activity calibrator, thus highlighting the high sensitivity of the digital autoradiography system. As expected, a significant and constant increase in the uptake of PSMA inhibitor was observed only in PSMA-positive tumours (LNCAP), while a decrease in the value of radioactivity was noted in other investigated organs as well as in the PSMA negative-tumours (PC3). These data were supported by the immunophenotypical characterization performed on both prostate cancer cell cultures and xenograft tumours. Indeed, no/rare PSMA-positive prostate cancer cells were observed.
Even though the distribution of radioactivity evaluated on whole mouse bodies by the digital autoradiography system cannot have the same sensitivity of micro-PET investigation, it allows an excellent space-time assessment of the biodistribution of a radiopharmaceutical. In particular, in this study, it was possible to follow the biodistribution of a PSMA inhibitor at three different time points, observing a progressive increase of radioactivity in the PSMA-positive tumour area.