Revisiting Pyronin Y, A Classical Photostable Xanthene Dye useful for Cost Effective Multimodal Fluorescence Microscopy

Abstract

In search of cost-effective alternatives for developing high-throughput imaging methods for NETosis, several classical histology dyes were revisited. It was found that the second component of the marketed premix Methyl Green-Pyronin Y (Gurr’s Certistain) has several properties compatible with this aim. Therefore, a simple purification method was developed, and the obtained Pyronin Y fraction, dried and redissolved in a biocompatible medium, was found to be photostable, selectively internalized by non-viable cells, and had spectroscopic features that make it compatible with many common fluorophores for multicolor imaging. Under certain conditions, Pyronin Y has a maximum absorption at 548nm, so it can be excited by 514nm and 561nm Laser lines, with an emission peak at 571nm. Experiments with human polymorphonuclear leukocytes (PMN) enzymatically induced to NETosis confirmed the usefulness of Pyronin Y in detecting early NETotic nuclei. By super-resolution microscopy, the compound enhances the visualization of nuclear subdomains, enabling the reduction of assay duration by detecting morphological alterations indicative of initial NETosis steps. Also, in chemically fixed resting PMN, azurophilic granules were stained by Pyronin Y, suggesting a previously unappreciated nucleic acid-independent staining mechanism. In addition, fluorescence lifetime imaging microscopy (FLIM) followed by time domain phasor analysis demonstrates the utility of the dye in differentiating between NETs released into the extracellular space and the genetic material still remaining inside the cell.