MERFISH measurements of a high-abundance RNA library in unexpanded U-2 OS samples. (a) Image of gel-embedded and cleared U-2 OS sample stained with encoding probes for 129 RNA species and a Cy5-labeled readout probe that detects one of the bits of the RNA barcodes at one focal plane of the z-scan. The yellow solid lines mark the segmentation boundaries separating neighboring cells, which do not necessarily represent the physical edges of cells. The yellow dashed lines mark the DAPI-stained cell nuclei. (b) Fluorescence images of all 8 rounds of two-color readout imaging for the orange boxed region in (a). Images were deconvolved and Gaussian filtered. Magenta and green represent the Cy5 and Alexa 750 channels, respectively. (c) The localizations of all decoded RNAs in (a) colored according to their measured binary barcodes. Decoded RNAs across all z-sections are displayed. The black solid lines mark the segmentation boundaries separating neighboring cells and the black dashed lines marks the DAPI-stained cell nuclei. (d) The average RNA copy numbers per cell for the 129 RNA species determined by MERFISH vs. the abundances as determined by RNA-seq. The Pearson correlation coefficient (r) between the log10 values of MERFISH-determined copy number per cell and RNA-seq determined FPKM value is 0.6. ~1,200 cells were measured in MERFISH experiments. (e) The average RNA copy numbers per cell determined by MERFISH (~1,200 cells) vs. those by smFISH (~1,000 cells per gene) for 12 of the 129 RNA species. The Pearson correlation coefficient (r) between the log10 values of MERFISH-determined and smFISH-determined copy numbers is 0.75. The average ratio of the copy number values determined by MERFISH to that determined by smFISH is 0.21 ± 0.04 (s.e.m., N = 12 RNA species) and the median ratio is 0.16. The scale bar in (a) represents 10 µm; the scale bars in (b) represent 1 µm.