Figure 2

bDNA amplification dramatically increases signal brightness without increasing spot size or variation in brightness from spot to spot. (a) Images of U-2 OS cells stained with smFISH probes to the filamin A (FLNA) mRNA. Left: an unamplified sample stained with a Cy5-labeled readout probe. Middle: the same image as on the left but with a 2x increased contrast to better illustrate the fluorescence signal. Right: a sample 5 × 5 amplified and stained with a Cy5-labeled readout probe. Scale bars, 10 µm. (b) Zoom in of white boxed region in (a). Scale bars, 2 µm. (c) The fold-increase in average brightness of individual FLNA mRNA spots after staining with amplifiers for different durations relative to the brightness observed for unamplified samples. (d) The fold-increase in average brightness of individual FLNA mRNA spots in 4 × 4 amplified, 5 × 5 amplified, and 9 × 9 amplified samples relative to the brightness observed for unamplified samples. The value reported for 5 × 5 amplification was reproduced from the 15 min measurement in (c). (e) The average FLNA mRNA spot sizes in unamplified, 4 × 4 amplified, 5 × 5 amplified, and 9 × 9 amplified cells. The width (full width at half maximum) was determined by Gaussian fitting of RNA spots. (f) The coefficient of variations for RNA spot brightness in unamplified, 4 × 4 amplified, 5 × 5 amplified and 9 × 9 amplified samples. Error bars in (c–f) represent standard deviation across three replicates.