Conceptual diagram of barcoding. (A) Schematic representation of the use of color groups to encode unique gene identities. Combinations shown are only two colors chosen from a total of four used throughout our experiments. A minimum of two colors is used to reduce the chance of single-color false positive signals. Each detected site is therefore representative of two or more independent hybridization events that are spatially colocalized, thus increasing fidelity. (B) Schema of the actual probe hybridizations at the transcription site leading to detection by barcode. The gene locus is represented (green) with several polymerases (blue) transcribing nascent messages (pink). A shotgun approach is used such that each specific probe may be of any of the colors in the barcode (red, yellow, magenta). The transcription site is shown below with hybridization data against the nuclear background (blue). Scale bar, 3 Ám. (C) An example of the signal readout interpreted by the transcription detection algorithm, showing the pixel intensities for the area of the transcription sites in all five color bandwidths. In this example, there is signal for each of the probe components used (red, yellow, and magenta) and only background levels for the color not in the barcode (purple).