MERFISH measurements of a high-abundance RNA library in expanded U-2 OS cells. (a) Schematic of the gel embedding and expansion for MERFISH imaging. (b) Image of an expanded 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. (c) Fluorescence images of all 8 rounds of two-color readout imaging for the orange boxed region in (b). (d) The localizations of all decoded RNAs in (b) colored according to their measured binary barcodes. Decoded RNAs across all z-sections are displayed. (e) The average RNA copy numbers per cell for the 129 RNA species determined by MERFISH (~750 cells) vs. the abundances determined by RNA-seq. The Pearson correlation coefficient (r) for the log10 values of RNA abundances is 0.83. (f) The average RNA copy numbers per cell for the 129 RNA species detected in one expanded sample (~750 cells) vs. a replicate sample (~250 cells). The Pearson correlation coefficient (r) is 0.94. (g) The average RNA copy numbers per cell for the 129 RNA species determined by expansion MERFISH (~1,000 cells) vs. those by MERFISH in unexpanded samples (~1,200 cells). The Pearson correlation coefficient (r) is 0.88. The copy numbers per cell detected in expanded samples were 7.5 ± 0.3 fold (average ± s.e.m., N = 129 RNA species) or 6.6 fold (median) of those detected in unexpanded samples. (h) The average RNA copy numbers per cell determined by MERFISH in expanded samples (~1,000 cells) vs. those by smFISH measurements in unexpanded samples (~1,000 cells per gene) for 12 of the 129 RNA species. The Pearson correlation coefficient (r) is 0.96. The average ratio between MERFISH and smFISH results is 1.05 ± 0.09 (average ± s.e.m., N = 12 RNA species) and the median ratio is 1.11. The scale bars in (b) and (c) represent 10 µm and 1 µm, respectively, with the grey scale bars representing the scales in the expanded sample and the orange scale bars representing the scales calculated back to sizes before expansion.