Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis

Abstract

Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

Figure 1

The lifetime image and phasor distribution of PY in DMEM ((A) and (B)) and RNA extracted from living NIH3T3 cells labeled with PY ((C) and (D)). The average lifetime for PY in solution and extracted RNA in this instance were determined to be 4.1 ns (red cursor) and 6.0 ns (green cursor), respectively. Cluster selection of a PY labeled cell (E) is presented in (F) (lifetime image) and (G) (phasor plot). The contributions of all three labeled components (PY in solution: red cursor; extracted RNA labeled with PY: green cursor; a PY labeled cell: purple cursor) are presented in the phasor plot in (I) with the selection of each cluster of pixels within the plot corresponding to the color-coded lifetime image in (H). Note the linear combination of extracted RNA labeled with PY and a cell labeled with PY.

Figure 2

The intensity image (A) and lifetime images ((B)–(E)) with phasors chosen through cluster selection in the polar plot pictured in (G), ((H): zoomed) with reference to the intensity image. Four distinct phasor clusters were selected which included most labeled cell regions. The average lifetime for each cluster selection was: red cursor 5.74 ns, purple cursor 5.65 ns, green cursor 7.03 ns and cyan cursor 4.68 ns. The phasor selected colored images of all four manually selected regions is presented in (F). In the images with color-coded phasor selections the intensity image is given using a grayscale.

Figure 3

Intensity image (A) and lifetime images ((B)–(F)) of RNA species in a single NIH3T3 cell labeled by PY. Cluster selection occurred through the phasor plot pictured in (H) ((I): zoomed) where color-coded cursors (yellow, red, green, purple and cyan) correspond to the images presented in (B)–(F). Five distinct phasor clusters were selected, outlining different regions of the cell (A). The average lifetime for each cluster selection was identified to be 4.77 ns (yellow cursor), 5.06 ns (red cursor), 6.13 ns (green cursor), 4.80 ns (purple cursor) and 3.68 ns (cyan cursor). The spatial distribution of the selected lifetimes is presented in (G). In the images with color-coded phasor selections the intensity image is given using a grayscale.

Figure 4

The intensity images ((A), (C) and (E)) and lifetime images ((B), (D) and (F)) of transcripts in a single NIH3T3 cell labeled by PY. The three images (A), (C) and (E) were taken at different planes about 1 μm apart and at different times, about 1 min apart. Cluster selection occurred through the phasor plot pictured in (G) ((H): zoomed) where color-coded cursors show the selection of average lifetimes in the images. Photostability was observed overall with only small alterations in the detected lifetime of most cellular compartments as a function of time (A)–(D). Small changes in focal plane (about 3 μm) still showed consistency in lifetime detection, for example in the nucleolus (C)–(F). The detected average lifetime values for each cursor were 4.51 ns (red), 4.56 ns (green), 4.18 ns (purple), 4.89 ns (cyan) and 4.09 ns (yellow).