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.