Active cyclin B1–Cdk1 kinase is located both in the cytoplasm and nucleus during prophase. HeLa cells coexpressing cytoplasmic and nuclear targeted cyclin B1–Cdk1 biosensors (fused to an NES and histone H2B, respectively) were recorded as cells entered mitosis (one image every 1 min 40 s). DIC (top), YFP emission (inverted grayscale; middle), and emission ratio (bottom) images are shown. Quantifications of the emission ratio in the whole cell, the nucleus, and the cytoplasm correspond to the cell displayed. Bar, 10 µm.