Fig. 1: STED-smFISH probe design and super-resolution imaging of mitochondrial mRNAs.

A Schematic representation of the STED-smFISH probe design and probe binding. From left to right: Both strands of the circular mtDNA are transcribed into polycistronic mRNAs, which are further processed to generate 11 mature mRNAs. The STED-smFISH probes consist of probe pairs (blue) that target a specific mRNA (magenta). Several probe pairs, depending on the length of the transcript, can bind simultaneously. Only when both probes of a pair are bound, the preamplifier (black) can bind. Multiple amplifier strands (orange) then bind to the preamplifier. The probe ‘tree’ is completed by the binding of the label probes (red), which are coupled to the fluorophore of choice (red stars). Specific probe pairs are available for all mRNAs (see Table 1). HSP: heavy strand promoter, LSP: light strand promoter. B Three-color confocal overview image of a U-2 OS cell labeled for the MT-ND1 mRNA (yellow), MT-CO3 mRNA (magenta), and MT-CYB mRNA (cyan). C Comparison between the STED and the confocal image at a higher magnification of the indicated area in (B). D Exemplary close-ups of spatially separated mRNAs (left) and mRNA clusters (right). E Full width half maximum (FWHM) of MT-ND1, MT-CO3, and MT-CYB mRNA spot sizes. N = 3, n = 171, mtRNA spots: 6674. F Quantification of the minimum pairwise distances between MT-ND1, MT-CO3, and MT-CYB mRNAs. N = 3, n = 70. Box: 25/75 percentile; whiskers: max/min without outliers; line: median; square: mean (E, F). Curved line: kernel density estimation representing the data point distribution (F). Source data are provided as a Source Data file. ‘N’ indicates biological replicates; ‘n’ indicates technical replicates. Scale bars: 5 µm (B), 1 µm (C), 50 nm (D).