Fig. 2

Accuracy and precision assessed by redundant detection. (A) Each target mRNA is detected using two probe sets, each initiating an orthogonal and spectrally distinct HCR amplifier (red channel, Alexa 647; green channel, Alexa 546). (B) Two-channel redundant detection of four target mRNAs: desma, Gt(desma-citrine) and elavl3 in whole-mount zebrafish embryos (fixed 26?hpf); and Acta2 in a whole-mount mouse embryo (fixed E9.5). Confocal microscopy: 0.7×0.7?µm pixels (desma, citrine and elavl3) or 0.07×0.07?µm pixels (Acta2). (C) Highly correlated normalized signal (Pearson correlation coefficient, r) for 2×2×2?µm voxels in the selected regions of B. Accuracy: linear with zero intercept. Precision: scatter around the line. (D) Scatter as a function of voxel size for desma. See section S2.1 in the supplementary material for control experiments testing for potential systematic penetration and crowding effects; see section S2.2 in the supplementary material for an examination of the effect of voxel size and probe set size on quantitative precision; and see section S2.3 in the supplementary material for additional data.