Fig. 2. Probing of intermolecular and intramolecular targets in the cytoplasm. (A) The detection of trans sequences. Both beta-(green) and gamma-(red) actin mRNA were detected simultaneously in the same cell with probes to their respective 3'-UTRs. The transcription sites for each isoform were visible in the nucleus. (B) Individual beta-and gamma-actin mRNAs segregate independently in the cytoplasm [statistical analysis of the frequency of measured separation distances in voxels (vo) (1 vo = 93 nm by 93 nm by 100 nm) between red and the nearest green signal is expressed as cumulative percent: 0 vo = 0.1%, 1 vo = 1.5%, 2 vo = 6.4%, 3 vo = 13.7%, 4 vo = 25.1%, 5 vo = 36.5%; n (red) = 685, n (green) = 459 (23)]. (C) The detection of cis-sequences. Two intramolecular regions of beta-actin mRNA are resolved with five different probes (12) targeted to the 3'-UTR (red) or the splice junction (SJ) sites (green). Beta-Actin mRNA molecules show nearest neighbor association of the 3'-UTR and the coding region. Frequency of separation distances in voxels: 0 vo = 2.4%, 1 vo = 15.8%, 2 vo = 32.9%, 3 vo = 59.1%, 4 vo = 72.6%, 5 vo = 87.2%; n (red) = 164, n (green) = 314. The 3'-UTR probes (red) are used to specifically detect beta-actin mRNA. The SJ probes for beta-actin (green) have homology with alpha-actin, accounting for more green than red signal (23). Respective red and green pairs had TFI values consistent with single -actin mRNA molecules. (D) Intramolecular measurement with three spectrally distinct probes hybridized to two -actin mRNAs. Two regions 631 bases (190 nm) apart were not resolved (CY5, blue; FITC, green); a third region, 1648 bases away (500 nm), was resolved (CY3, red). The colocalized voxels of the blue and green images are turned white. The distance between red and blue maxima is estimated to be 487 nm, consistent with a linear RNA. The schematic depicts the locations of the probes. In (A), bar = 5 µm; in (B) and (C), bar = 93 nm; and in (D), bar = 187 nm.