Fig. 3 |. Quantification of single-transcript intensity units in multiple channels.

a, The single-dot transcript intensity distribution (top) can be obtained by subtracting a background distribution obtained without FISH probes (middle, histogram of n = 1,953) from the foreground plus background image obtained with FISH probes (bottom, histogram of n = 1,054). b, Applying this procedure to each channel and fitting the resulting distributions to a Poisson distribution provides the three single-transcript intensity units (indicated). c, The measured single-transcript intensity units in multiple channels are verified via dot-co-localization (Supplementary Fig. 1d). Different methods generate similar single-molecule fluorescence units. Color is labeled as in Fig. 2a. d, The properties of background dots (gray, n = 5,000) and identified true FISH dots (pink, n = 10,000). Note overlap of these properties. See more dot-fitting properties in Supplementary Note 2. e, The measured fluorescent intensity for staining of SDHA gene is proportional to the number of smFISH probes included (indicated numbers). A negative control (brown) uses a fixed number of probes and displays a relatively fixed fluorescence intensity. This indicates that intensity scales linearly with the number of probes. The data are for n = 3,000 and n = 200dots of SDHA and control staining, respectively. The measure of center is the mean. Error bars represent standard deviation. Source data for panels d and e are available online.