Fig. 1.

Approaches to improve the measurement throughput of MERFISH. (A) Simplified schematic of a MERFISH readout protocol. Target RNAs are stained with encoding probes that contain a barcode comprising a combination of readout sequences unique to each RNA species. The barcode then is identified through successive rounds of smFISH, each with a readout probe complementary to one readout sequence. A registered stack of smFISH images for each sample produces an ensemble of fluorescence spots with on/off patterns that define binary barcodes (“1” represents fluorescent signal on, and “0” represents fluorescent signal off) which allow individual RNA species to be identified. A more detailed hybridization and imaging procedure is shown in Fig. S1. (B) The time required to perform a MERFISH experiment for a given sample area for the published protocol (18, 23) that uses photobleaching to remove smFISH signal (red line), a modified protocol without photobleaching (purple line), a modified protocol without photobleaching and a larger FOV (green line), and a modified protocol without photobleaching, a large FOV, and two-color imaging (blue line).