To maximize fruit yield of tomatoes cultivated in a controlled, closed system such as a greenhouse or a plant factory at a limited cost, it is important to raise the translocation rate of fixed carbon to fruits by tuning the cultivation conditions. Elevation of atmospheric CO₂ concentration is a good candidate; however, it is technically difficult to evaluate the effect on fruit growth by comparing different individuals in different CO₂ conditions because of large inter-individual variations. In this study, we employed a positron-emitting tracer imaging system (PETIS), which is a live-imaging technology for plant studies, and a short-lived radioisotope ¹¹C to quantitatively analyze immediate responses of carbon fixation and translocation in tomatoes in elevated CO₂ conditions. We also developed a closed cultivation system to feed a test plant with CO₂ at concentrations of 400, 1,500 and 3,000 ppm and a pulse of ¹¹CO₂. As a result, we obtained serial images of ¹¹C fixation by leaves and subsequent translocation into fruits. Carbon fixation was enhanced steadily by increasing the CO₂ concentration, but the amount translocated into fruits saturated at 1,500 ppm on average. The translocation rate had larger inter-individual variation and showed less consistent responses to external CO₂ conditions compared with carbon fixation. Our experimental system was demonstrated to be a valuable tool for the optimization of closed cultivation systems because it can trace the responses of carbon translocation in each individual, which are otherwise usually masked by inter-individual variation.