Current optical and electron microscopy techniques have limitations when it comes to examining the dynamic properties of living microorganisms at minute scales. These challenges arise largely from complex sample preparation requirements, as well as issues with resolution and imaging speed. To tackle this problem, a research team has refined high-speed atomic force microscopy (HS-AFM) technology, allowing them to obtain nanoscale physical and mechanical data more efficiently, adapting it for live specimens. They developed a new approach called high-speed in-line force mapping (HS-iFM), which has successfully been used to analyze the mechanical traits and features of dividing *E. coli* cells, achieving both high resolution and speed.
Optical and electron microscopy each come with their own set of challenges. While light microscopy struggles to discern increasingly smaller details, electron microscopy can resolve tiny structures, yet it requires extensive sample preparation, which unfortunately eradicates live samples.
Atomic force microscopy (AFM) was initially designed to evaluate the physical and mechanical properties of materials at exceptionally high resolutions. However, its imaging speed, which can take several minutes per frame, is insufficient for capturing vital data related to living biological samples. On the other hand, high-speed AFM (HS-AFM) offers improved speed but lacks the capability to measure mechanical properties. Acknowledging the potential of microscopy for investigating large molecules and microorganisms, researchers from the National Institutes of Natural Sciences (NINS) and Nagoya University devised a novel technique, HS-iFM, to gather dynamic mechanical force measurements at a speed and resolution suitable for living biological samples. The team, consisting of Christian Ganser, Shigetaka Nishiguchi, Feng-Yueh Chan, and Takayuki Uchihashi, opted to start their explorations with the well-studied bacterium *Escherichia coli*.
The researchers shared their findings in the January 29 edition of *Science Advances*.
“Although studying ‘static’ samples, like non-living bacteria, provides useful information, observing live organisms allows us to directly track their life changes over time, which is possible with our technique. *Escherichia coli* serve as an excellent model due to their extensive research background. However, the dynamic mechanical changes occurring at the nanoscale level in *E. coli* remain poorly understood,” stated Christian Ganser, an assistant professor at the Exploratory Research Center on Life and Living Systems (ExCELLS) within NINS in Okazaki, Japan.
Throughout the division of *E. coli* cells, the research team noticed an increase in the mechanical stiffness at the division site. They propose that this increase may stem from localized membrane tension and thickening of the cell wall. “The division site becomes substantially stiffer than the surrounding areas, suggesting significant internal stresses required to change the membrane and separate the cells,” added Ganser.
As the cell divided, the membrane created visible connections, or bridges, between the two daughter cells that would stretch and eventually snap. The formation and breaking of these bridges had an average duration of 242 seconds ± 99 seconds (mean ± standard deviation) and occurred on seven separate instances. A supplementary video detailing the entire division process is available along with the online publication of the study.
Moreover, the team identified a weakness, measuring under 100 nm in diameter, in a dividing *E. coli* cell, which led to a rupture, causing loss of internal pressure and cell death. Notably, the bursting cell affected both daughter cells, indicating that internal separation had not been fully achieved. This finding suggests the potential of HS-iFM in timing various steps during the cell division of *E. coli* and similar bacteria.
HS-iFM enables researchers to assess both high-resolution topography and the mechanical properties of membranes. During the division of a living *E. coli* cell, the team observed temporary membrane holes that appeared to close, reform, and shift across the membrane. They propose that these dynamic hole structures may relate to the development of outer membrane vesicles, which tend to be generated more frequently during cell division and in the new cell walls forming between the daughter cells. Alternatively, these pores could represent outer-membrane protein complexes. However, the measured pore diameter of 34.7 nm ± 11.8 nm (1 nm = 1 x 10-9 m) is notably larger than previously reported protein complex sizes, which are around 8 nm.
The research team recognizes the significant promise held by the HS-iFM technique to explore a variety of biological samples and organisms, including *E. coli*. “In the future, we plan to apply our method to investigate the dynamic and localized effects of external stimuli, such as antibiotics, on the nanomechanical attributes of living bacterial membranes,” shared Ganser. They also envision using HS-iFM to explore the temporary nanomechanical characteristics of polymers. Ideally, the team aims to enhance the speed and resolution of this technique to visually capture the mechanical properties of tiny molecules, including individual proteins.
The co-authors of this research paper include Shigetaka Nishiguchi from the Exploratory Research Center on Life and Living Systems at the National Institutes of Natural Sciences in Okazaki, Japan; Feng-Yueh Chan from the Department of Physics at Nagoya University; and Takayuki Uchihashi from both the Exploratory Research Center on Life and Living Systems at the National Institutes of Natural Sciences and the Department of Physics at Nagoya University.
This research received support from JSPS KAKENHI Grant Number JP23H04874 under the Grant-in-Aid for Transformative Research Areas “Materials Science of Meso-Hierarchy,” as well as 24K01309 and 22K18943; from JST, CREST Grant Number JPMJCR21L2, Japan; and through MEXT’s Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence Program (CURE) Grant Number JPMXP1323015482.
