Correlation of Live Imaging and Fixed-Expansion Microscopy to study intracellular pathogens
This study introduces a LiFEx workflow that correlates live-cell imaging with adapted expansion microscopy to overcome bacterial expansion limitations and reveal dynamic host-pathogen interactions, specifically linking T6SS-5 assembly to rapid membrane alterations during *Burkholderia thailandensis* infection.
Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer
To see the invisible machinery of life, scientists often have to shrink their tools or build them from scratch. For decades, the limit of what a light microscope could show was set by the physics of light itself; tiny structures smaller than half a wavelength of light would blur together, hiding the intricate details of cells and the bacteria that live inside them. Expansion microscopy emerged as a clever workaround to this problem. Instead of building a more powerful lens, researchers found a way to physically stretch the specimen itself. By embedding a sample in a special gel that swells when soaked in water, they can pull the biological structures apart, making them larger and easier to see with standard microscopes. This technique has revolutionized how we view the architecture of cells, but it has hit a wall when applied to bacteria. These microscopic invaders are wrapped in a tough, protective shell that resists stretching, often causing them to remain small and distorted while the surrounding host cell swells up. Understanding how bacteria move and spread between human cells requires seeing these tiny structures clearly, yet the very armor that protects the bacteria also hides them from view.
A team of researchers at the University of Basel has now adapted this stretching technique to peer inside human cells infected with Burkholderia thailandensis, a bacterium that serves as a model for a more dangerous pathogen. They wanted to understand how these bacteria move from one cell to another, a process that involves pushing against the cell membrane to form a bridge into a neighbor. The challenge was that while the human cells in their experiments expanded nearly nine times their original size, the bacteria inside them refused to stretch as much, remaining shrunken and uneven. The researchers set out to figure out why the bacteria were resisting the expansion and to develop a way to link what they saw in the stretched, frozen samples back to the living, moving events that happened just moments before.
The team first tackled the question of what was holding the bacteria back. They suspected the bacterial cell wall, a rigid mesh of sugar and protein that acts as a shield, might be the culprit. In their experiments, they tried removing this wall using enzymes before stretching the sample. Surprisingly, removing the wall did not make the bacteria expand fully; they still remained smaller than the surrounding human cells. They also tested whether the outer membrane of the bacteria was too tight or if the proteins inside were too tightly packed to allow for stretching. Even when they used stronger enzymes to digest the proteins or chemicals to loosen the outer membrane, the bacteria still did not stretch uniformly. The only factor that seemed to make a significant difference was how the cells were preserved before the experiment began. The researchers found that the specific mixture of chemicals used to fix the cells—essentially to freeze them in place—determined whether the bacteria would stretch evenly or remain distorted. This discovery suggests that the way scientists prepare their samples is just as critical as the stretching process itself.
Despite the bacteria not stretching perfectly, the new method allowed the team to see details of the infection that were previously impossible to resolve. They observed the bacteria pushing against the human cell membrane to create long, thin protrusions that reach into neighboring cells. In some cases, these protrusions were smooth and continuous, but in others, the membrane appeared broken or fragmented. The researchers could see that the bacteria were using a specialized molecular machine, known as a secretion system, to punch holes in these membranes and escape into the next cell. However, because the standard method only shows a static snapshot after the sample is fixed, they could not tell exactly when the membrane broke relative to the moment the bacteria fired their molecular machine.
To solve this, the team developed a new workflow they called LiFEx, which stands for Live-to-Fixed Expansion microscopy. This approach allowed them to watch the bacteria move in real time, record the exact moment a specific event occurred, and then immediately fix the sample in place without moving it. They could then process that same tiny area through their expansion protocol and look at it again under a high-powered microscope. By comparing the live video with the stretched, high-resolution image, they could measure the time delay between the bacteria assembling their molecular weapon and the actual rupture of the cell membrane. They found that the membrane did not break instantly; instead, it took several minutes for the structural damage to become visible after the bacteria fired their system.
This work provides a clearer picture of how intracellular pathogens navigate their environment. The researchers demonstrated that while the bacterial cell wall is a factor, it is not the sole barrier to seeing these tiny structures clearly, and that the chemical conditions used to preserve the sample play a major role in the final image quality. More importantly, by combining live observation with high-resolution structural analysis, they revealed the precise timing of how these bacteria breach the defenses of their host cells. The study does not claim to have solved the problem of bacterial expansion entirely, as the bacteria still did not stretch as much as the human cells, but it offers a robust method to study the dynamic interactions between pathogens and their hosts with a level of detail that was previously out of reach.
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