Non-invasive Quantification of Cell Wall Porosity by Fluorescence Quenching Microscopy

All bacteria, fungi and plant cells are surrounded by a cell wall. This complex network of polysaccharides and glycoproteins provides mechanical support, defines cell shape, controls cell growth and influences the exchange of substances between the cell and its surroundings. Despite its name, the cell wall is a flexible, dynamic structure. However, due to the lack of non-invasive methods to probe the structure, relatively little is known about the synthesis and dynamic remodeling of cell walls. Here, we describe a non-invasive method that quantifies a key physiological parameter of cell walls, the porosity, i.e., the size of spaces between cell wall components. This method measures the porosity-dependent decrease of the plasma membrane-localized fluorescent dye FM4-64 in the presence of the extracellular quencher Trypan blue. This method is applied to bacteria, fungi and plant cell walls to detect dynamic changes of porosity in response to environmental cues.