Background

Bacterial, fungal and plant cells are surrounded by a cell wall which has a multitude of different functions, including, defining size and shape, and controlling the exchange of substances within the environment. A key feature of cell walls is their flexibility. Long polysaccharides form a strong network and their structure is frequently adjusted, for example to allow for cell elongation, or to counter pathogen attack. In addition, cell wall porosity allows for molecular movement between the wall’s various components. Cell wall porosity is also a reliable indicator of digestibility and saccharification efficiency of cell wall material (Himmel et al., 2007; Ding et al., 2012; Tavares et al., 2015) and has been linked to anti-fungal drug uptake (Liu et al., 2019). Current methods for measuring cell wall porosity are based on relatively invasive methods, such as transmission electron microscopy (TEM) or field-emission scanning electron microscopy (FESEM) (Sugimoto et al., 2000; Xiao et al., 2016; Zheng et al., 2017), which alter the original cell wall structure and may lead to artifacts. Another method, cryo-FESEM, is less invasive but the resolution is limited to 20 nm (Derksen et al., 2011). Pore size distributions at sub-nanometer resolution can be obtained by gas adsorption, but this method also requires harsh sample pretreatment (Adani et al., 2011). Instead of determining pore sizes directly, other methods measure the capacity for molecular movement within the wall, which can be compatible with live-cell analysis. For example, the relative porosity of yeast cell walls assesses the measuring the effect of different-sized polycations on the leakage of UV-absorbing intracellular compounds (De Nobel et al., 1990). Similarly, the effect of fluorescent quenchers with different hydrodynamic radius on the autofluorescence of lignin can be measured to gain insight on differences in porosity between wood samples (Donaldson et al., 2015).

The method presented here makes use of the ability of quenchers to diffuse through the cell wall to determine relative porosity. In contrast to the approaches described above, this method works on living cells and can be applied to all accessible tissues and all organisms. It measures the fluorescence intensity of the dye FM4-64 located at the plasma membrane in the presence of extracellular quenchers. The quenching effect has been shown to depend on cell wall porosity (Liu et al., 2019). The method has been successfully used on bacteria, fungi and plants, for example to determine changes in wall porosity during drought-induced cell elongation in the model plant Arabidopsis (Liu et al., 2019).

The method described below uses Trypan blue as a quencher. In comparison with other quenchers, Trypan blue has a higher dynamic range (Liu et al., 2019), which results in more reliable results when analyzing cell walls with low porosity. Furthermore, Trypan blue is widely available, doesn’t require co-factors and is inexpensive. The protocol for yeast cells can be quickly adapted to any other microorganism, by choosing the right culture medium and growth conditions. Similarly, the protocols for Arabidopsis roots and Maize leaf samples can be adapted to other plant species. For root samples, however, growth on agar plates is recommended instead of cultivation on soil, because it can be difficult to clean all soil particles without damaging the root surface.