Figure 5. Imaging RNA in yeast cells with Spinach and IMAGEtag reporters.

A) Yeast cells expressing control RNA, SPN1A or 2xSPN2A were incubated at 30°C with DFHBI or PFP-DFHBI and fluorescence and DIC images taken after 60 min, B) The fluorescence images in A, and others from the same experiment, were quantified for fluorescence per cell using ImageJ. The results are shown for each ligand normalized to the average fluorescence per cell of the control RNA-expressing cells. Images of field as higher resolution are shown in Fig. S1 and Fig. S2. C) Representative images of cells (63x objective with 3.5X zoom in) from the experiment in A to demonstrate that the high background signal is not due to vacuolar uptake of the ligands. D) Images of yeast cells expressing GAL1 promoter-driven 6XPDC IMAGEtags taken 99 min after the promoter was induced by the addition of 2% galactose to cells that had previously been grown for 12 h in 2% raffinose to reach 0.7 OD⁶⁰⁰. The images for Cy3-PDC only, Cy5-PDC only, or Cy3-PDC, Cy5-PDC are of cells that have been incubated with either one or the other PDC ligand or the ligand pair (Cy3-PDC, Cy5-PDC) that interacts in FRET. Images were taken in the Cy3, Cy5 or FRET channels. The DIC is of the field shown for the ligand pair and is representative of all fields in this experiment. E) FRET efficiencies from the experiment in D determined from images taken each 1 or 2 min after induction were quantified for 8 cells expressing control RNA and for 12 cells expressing 6xPDC IMAGEtags and averaged for each group. The average FRET/cell of the IMAGEtag expressing group was divided by the average FRET/cell of the control RNA expressing group and plotted as a function of time after the addition of galactose. F) The chemical structure of Cy3-PDC. Cy5-PDC is identical in structure with the exception that Cy5 replaces Cy3.