Imageem c9100 14

Radioluminescence imaging was performed using a bioluminescence microscope (LV200, Olympus) outfitted with a 40×/1.3 NA oil objective (UPLFLN40XO, Olympus), and a deep-cooled electron-multiplying charge-coupled device (EM-CCD; ImageEM C9100-14, Hamamatsu). All samples were imaged using 4×4 binning and an electron-multiplication gain of 1200. Fluorescence imaging was performed on Leica DM6000B microcope using a Hamamatsu C11440 fluorescence camera and a Leica DFC450 brightfield camera, with 20× magnification and an exposure time of 4 seconds.

Prior to imaging, cells were fasted in glucose-free DMEM or RPMI medium supplemented with 10% fetal bovine serum for 45 minutes at 37°C and 5% CO₂. Subsequently, FDG (20 MBq/mL) was introduced into the dish for uptake by cells for 45 min (37°C and 5% CO₂). The cells were then washed three times with DMEM medium supplemented with 10% fetal bovine serum to remove residual FDG. Following these preparations, the cells were imaged using RLM (Fig. S1 A) using a CdWO₄ scintillator (1 cm×1 cm×0.5 mm, 2 sides polished, MTI Inc). Images of individual ionization tracks were serially acquired using an EM-CCD (Hamamatsu ImageEM C9100–14) with maximum gain, 50–200 ms integration time, and 4×4 binning. The integration time was chosen to average about 10 decay events per frame. Reconstructed RLM images were obtained by our methodology called optical reconstruction of the beta-ionization track (ORBIT) as previously described in detail (24 (link)). The methodology is summarized in the supporting material (Fig. S1). Region-of-interest (ROI) analysis of single cells was performed on the reconstructed images by measuring the total number of counts inside circular ROIs placed manually on individual cells, using the corresponding brightfield and bioluminescence images as a reference.