Prostate cancer (PCa) patients may develop local recurrences and metastatic spread after primary treatment. A general indicator of possible relapse is an increasing concentration of prostate-specific antigen (PSA) in the blood. PSA is generally regarded as a good biomarker for disease progression in primary PCa, and it is a well-studied surrogate of tumor burden [
1‐
4]. However, at an early PCa recurrence or microscopic metastatic spread, PSA alone can seldom be used for staging and is generally associated with relatively low levels of serum PSA concentrations (< 0.2 ng/ml). If the PCa recurrence is diagnosed at an early stage, the patient can still be treated with curative intentions. A recent study showed that patients with biochemical PCa recurrence and treated with salvage radiation therapy (SRT) have a long term disease-free rate which rapidly declines with increasing levels of PSA at treatment start [
5]. Further, another study showed that about half of the PCa patients treated with SRT are likely to suffer again a biochemical failure [
6]. These adverse therapeutics results may correspond to patients with disseminated disease, which should have been treated with systemic therapy instead of—or in combination with—SRT, which is more successful for focal recurrences. These patients may even become more symptomatic after SRT. Hence, the proper imaging diagnostic tool to discriminate patients with local recurrence from advanced metastatic PCa is essential to select the most adequate treatment strategy based on the spread and anatomical location of the disease. However, to date, there is no gold standard diagnostic imaging technique for this patient group. At low PSA levels, conventional imaging diagnostic techniques, like ultrasound, magnetic resonance, and computed tomography, have shown limitations at discriminating patients with local recurrence from those with disseminated disease [
7]. Deep biopsies are, on the other hand, difficult to correlate with imaging, have difficult histological interpretation and need skills to be successfully accomplished [
8,
9]. Recently, positron emission tomography/computer tomography (PET/CT) imaging of the Gallium-68-labeled prostate-specific membrane antigen (Ga68-PSMA) has emerged as a potential gold standard to monitor PCa recurrence/metastatic spread even at low PSA levels [
10,
11]. The literature on the topic revealed that, from all image modalities, Ga68-PSMA-PET/CT is to date the most accurate test for patient follow-up and staging. Ga68-PSMA-PET/CT outperforms also the detection rates of other PET tracers commonly used for PCa-like C11-Acetate and C11-Choline, even at low PSA [
12]. Ga68-PSMA-PET/CT images have recently been used to deliver SRT with dose escalation to focal and solitary Ga68-PSMA uptakes in the prostate bed and pelvic nodes [
13]. In such approach, good knowledge on the accuracy of Ga68-PSMA-PET to establish micro-metastatic involvement is therefore essential for patient selection. Still, there is no clear consensus on the limitations of this technique [
14]. This may be due to the lack of a gold standard to which it can be compared against at low PSA and a lack of studies on the limitations of the Ga68-PSMA-PET/CT-imaging technique at microscopic stage. Modern PET cameras have high sensitivity, but at very low cellular concentration levels, the limiting physical factors inherent to the PET technique [
15‐
17] as well as the specific biological and pharmacological properties of the Ga68-PSMA tracer hamper tumor imaging performance. Phantom experiments characterizing PET tracer detectability are generally useful only to characterize one or a few of these factors at a time. Put together, phantom-derived PET scanner performance, in terms of minimum detectable activity for Ga68-PSMA tracer, may not reflect the true in vivo realization. The aim of this study was to determine the fundamental limits of Ga68-PSMA-PET/CT in localizing small prostate cancer cell deposits, by taking into consideration the summative effects of the physical, biological, and pharmacological factors in a single set of experiments. Additionally, the quantitative properties of PCa imaging with Ga68-PSMA-PET/CT were also investigated.