In a living cell, gene expression—the transcription of DNA to messenger RNA followed by translation to protein—occurs stochastically due to the fact that DNA are single molecules in individual cells. Recent advances in single-molecule imaging in living cells allow quantitative and system-wide descriptions of gene expression and regulation. It was found that low probability events of single molecules can have important biological consequences, such as the change of a cellular phenotype. Meanwhile, recent advances in high throughput DNA sequencing have allowed sequencing of the genome of a single human cell. The combination of single molecule and single cell imaging and sequencing offers exciting possibilities for biology.