Concentration,cellular exposure and specificity of organelle selective fluorescent dyes in fungal cell biology

This technical focus article discusses the importance of concentration, cellular exposure and specificity for the application of organelle selective fluorescent dyes in fungi using DNA, membrane and cell wall stains as examples. Nonetheless, the presented considerations are generally applicable to all fluorescent dyes applied to living cells.The association of a fluorescent dye with its target molecule generally impairs molecule and consequently organelle function. Effective dye concentration, cellular exposure time and specificity to the target molecule are key factors that influence the biocompatibility of any fluorescent dye. Prominent molecules frequently used as fluorescent staining targets in fungal cell biology are: (i) DNA for nuclear labelling, (ii) α-/β-glucans and chitin for cell wall labelling, and (iii) phospholipids for plasma membrane and endomembrane labelling. In combination with live-cell imaging settings that reduce light stress, i.e. excitation intensities and exposure times set to the minimum that still achieves good signal-to-noise ratios, is the low dosage application of fluorescent markers as so called “vital dyes” essential for visualising cellular processes in an artefact-free fashion.