(a) Plate layout with 3 types of culture medium tested on a p96-well plate. (b) Example of cells imaged by widefield time-lapse microscopy for at least 8 hours. After more than 4 hours of acquisition, HeLa cells clearly required serum to stay alive and spread correctly on the support. After 8 hours of acquisition on the setup in the Fluorobrite+Glutamax+serum medium, a similar number of cells and identical shapes were obtained as compared to classical medium (DMEM+Glutamax+SVF), validating the use of Fluorobrite medium as an imaging medium for our living cells experiments. (c) No difference in background fluorescence (yellow boxes, quantification on right) or single molecule intensity (green boxes) was noticed between Fluorobrite+Glutamax+serum medium and PBS.