Abstract Most cells adapt to their environment by switching combinations of genes on and off through a complex interplay of transcription factor proteins (TFs). The mechanisms by which TFs respond to signals, move into the nucleus and find specific binding sites in target genes is still largely unknown. Single-molecule fluorescence microscopes, which can image single TFs in live cells, have begun to elucidate the problem. Here, we show that different environmental signals, in this case carbon sources, yield a unique single-molecule fluorescence pattern of foci of a key metabolic regulating transcription factor, Mig1, in the nucleus of the budding yeast, Saccharomyces cerevisiae . This pattern serves as a ‘barcode’ of the gene regulatory state of the cells which can be correlated with cell growth characteristics and other biological function. Highlights Single-molecule microscopy of transcription factors in live yeast Barcoding single-molecule nuclear fluorescence Correlation with cell growth characteristics Growth in different carbon sources

Correlative single-molecule fluorescence barcoding of gene regulation in Saccharomyces cerevisiae