Spectral phasor plot for hyperspectral imaging of acridine orange photophysics within the cell

Abstract

AbstractAcridine Orange (AO) has rich photophysics that has been exploited to study its interaction with RNA and DNA. Traditionally AO was used to stain cells differentiating nucleus and cytoplasm due to the differences in DNA and RNA differential composition. AO cell labeling shows complex behavior depending on several parameters, such as concentration, pH, etc. Such complex photophysics will benefit from a model-free analysis such as the spectral phasor (SP) for hyperspectral imaging. The SP linear combination and reciprocity principles allow us to understand better the AO spectral shift associated with its exploration of the different molecular environments in cells. Using this approach, we identified the SP fingerprints for RNA and DNA for in vitro experiments, which remain invariant for fixed and live cells. However, the SP analysis of the AO fluorescence from live cells shows the occurrence of a third spectral component at 640 nm. The linear properties of the SP analysis enable quantification of the AO bound to RNA, DNA, and a third component. By co-labeling with AO and LysoTracker, we associated part of these subcellular structures with acidic compartments. Nonetheless, we could differentiate AO self-interactions from its binding to RNA within cytoplasmic compartments by use of the SP analysis. This work establishes the use of SP for hyperspectral imaging of AO for the first time, expanding our knowledge of its complex photophysics and promoting further application for in vivo cell imaging.