Spectral Phasor Analysis for Hyperspectral Imaging to elucidate Acridine Orange Photophysics in Cells

Acridine Orange (AO) exhibits complex photophysical properties that have been utilized to study its interactions with RNA and DNA. Traditionally, AO has been used to stain cells, distinguishing nucleus and cytoplasm based on differences in DNA and RNA composition. Its labeling behavior varies depending on factors such as concentration and pH. AO fluorescence is rich and complex; therefore, acquiring the full spectrum or the fluorescence lifetime is beneficial for understanding its intricate photophysics. Nevertheless, handling 4D datasets can be challenging and often relies on sophisticated or highly parameterized models for their analysis. Model-free approaches, such as spectral phasor (SP) analysis for hyperspectral imaging (HSI), are well suited to navigating these analytical challenges. Using SPs principles of linear combination and reciprocity, we identified spectral fingerprints of RNA and DNA in in vitro experiments that remain consistent in both live and fixed cells. Interestingly, SP analysis of AO fluorescence in live cells revealed a third spectral component at 640 nm. The linear properties of SP analysis enabled the quantification of AO bound to RNA, DNA, and the third component. Co-labeling with AO and LysoTracker suggested that some of these subcellular structures are associated with acidic compartments. Moreover, SP analysis distinguished AO self-interactions from its RNA binding in cytoplasmic compartments. This study establishes SP analysis as a robust tool for hyperspectral imaging of AO, thereby enhancing our understanding of its intricate photophysics and unlocking new opportunities for advanced in vivo cell imaging applications.