Abstract
We show that the spectral phasor approach of the fluorescent dye Pyronin Y (PY) can be used to identify specific RNA subspecies of ribonuclear proteins complexes in live cells. We applied spectral phasors to isolate intracellular RNA species with similar spectral properties. We identified at least 4 different PY labeled species in live cells and further spatially mapped their presence at the pixel level. Most notable were transcripts in the nucleoli which were spectrally similar to RNA clusters in the cytoplasm. We propose that these species represent ribosomal RNA and clustered ribonucleoprotein complexes. Further, we observed within this cluster Cajal bodies in the proximity of the nucleolus. In addition, transcripts in the cytoplasm undertook a filamentous morphology composed of multiple puncti structures which individually localized along and close to mitochondria but were distinct from mitochondria.
Highlights
The fluorescent probe Pyronin Y (PY) is an environment sensitive probe which has been used to target cell structures including RNA, DNA and organelles
We show that the spectral phasor approach of the fluorescent dye Pyronin Y (PY) can be used to identify specific RNA subspecies of ribonuclear proteins complexes in live cells
We identified at least 4 different PY labeled species in live cells and further spatially mapped their presence at the pixel level
Summary
The fluorescent probe Pyronin Y (PY) is an environment sensitive probe which has been used to target cell structures including RNA, DNA and organelles. The aim of this study is to determine whether there are sufficient differences in the spectral properties of PY that can be use to determine which nucleic acid subtype PY is bound in the cell environment This is of relevance in the cytoplasm and nucleus where multiple RNA subtypes may be present [5] and distinction of these PY labeled species based on intensity only is unfeasible. In a microscopy fluorescence image of a cell, PY may bind to the various transcript subtypes resulting in the inability to distinguish which RNA the probe is bound to Overall this means the apparent uniform labeling of cytoplasmic transcripts as well as labeled transcripts in the nucleolus are generically attributed to dsRNA. As a consequence there is no definitive way to identify which species of RNA are positioned where in the cell, or the number of species present in the analysis
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