Novel microscopy technique reveals dynamic stiffness of E. coli membrane

Light and electron microscopy each have their distinct limitations – with light it becomes increasingly difficult to resolve smaller and smaller features while electron microscopy can resolve small structures but its samples must be meticulously prepared which will kill any live specimens.

And then there is atomic force microscopy (AFM) which is a technique originally developed to assess the physical and mechanical properties of materials at extremely high resolutions. Here imaging speeds are not fast enough – several minutes per frame –to capture relevant data for living biological samples. By contrast, a further method, high-speed AFM (HS-AFM), is far faster but cannot measure mechanical properties.

Understanding the potential of this type of microscopy for the analysis of large molecules and microorganisms, researchers from the National Institutes of Natural Sciences (NINS) and Nagoya University in Japan created a new technique – high-speed in-line force mapping (HS-iFM) – to acquire dynamic, mechanical force measurements at the speed and resolution required for living biological samples.

The research team chose the common bacteria Escherichia coli ( E. Coli) to be its first subject to study in unprecedented detail.

“Observing living organisms [and] directly follow changes in the course of its life… …became possible with our technique. E. coli bacteria are great to demonstrate such effects because they are well studied. Despite this, the mechanical changes occuring in E. coli at the nanoscale level… have remained elusive,” said Dr. Christian Ganser, assistant professor at the Exploratory Research Center on Life and Living Systems (ExCELLS) in the NINS in Okazaki, Japan.

During cell division in E. coli, the researchers observed an increased mechanical stiffening at the site of cell division. The researchers hypothesised this stiffening could be due to localised membrane tension and local cell wall thickening.

“The division site becomes much stiffer than the surrounding cell, hinting at large internal stresses that are needed to deform the membrane and separate the cells,” said Ganser.

During division, the membrane also formed visible bridges between the two daughter cells that stretched and eventually broke. The creation and eventual breakage of these bridges lasted an average of 242s ± 99s (mean ± standard deviation) and was observed seven times.

The team also observed a weak spot, less than 100nm in diameter, in a dividing E. coli cell that ruptured, causing cell depressurisation and death. Interestingly, the bursting cell caused depressurisation of both left and right daughter cells, indicating that, internally, the cells had not completely separated. This observation suggests that HS-iFM may be useful in determining the timing of different steps during the division of E. coli and other bacteria.

HS-iFM allowed the researchers to measure both high resolution topography and membrane mechanical properties. During division of a living E. coli cell, the researchers observed visible membrane holes that close and reform again and diffuse across the membrane.

The team hypothesised that these dynamic hole structures may be related to the formation of outer membrane vesicles, which are reported to be more frequent during cell division and around the new cell walls that develop between daughter cells during bacterial cell division. The pores could also be outer-membrane protein complexes located on the bacteria surface, but the measured pore diameter of 34.7nm ± 11.8nm (1nm = 1 x 10^-9 m) is significantly larger than previous reports of protein complex diameter, which is approximately 8nm.

The research team acknowledges the great potential of the HS-iFM technique for studying a wide variety of biological samples and organisms, including E. coli.

“In the future, we will use our technique to study the dynamic and localised effect of external stimuli, such as antibiotics, to the nanomechanical properties of membranes of living bacteria,” said Ganser.

The researchers also envision using HS-iFM to study the transient nanomechanical properties of polymers. Ideally, the team will increase the speed and resolution of the technique to visually capture the mechanical properties of molecules as small as individual proteins.

For further reading please visit: https://www.science.org/doi/10.1126/sciadv.ads3010