A new microscope capable of capturing live, detailed imaging of biological processes — down to the movement of protein complexes — has been assembled. “Our ambition is to be able to visualize biochemical processes occurring mainly in liquid with unprecedent resolution,” explained Elena Macías-Sánchez, researcher at Radboud University Medical Center (Radboudumc) and the University of Granada.

Most synthetic and biological processes take place in liquids, which makes it much harder for scientists to visualize them in detail “Liquids pose an inherent problem for high-resolution electron microscopy, because the column vacuum required for electron transmission causes the water molecules to evaporate, collapsing the sample,” said Macías-Sánchez.

Most techniques can only capture snapshots of the object being studied, offering limited insights and no information on its past or future behavior.

“Traditional methods could only capture biological processes as static images or in lower resolution,” said Nico Sommerdijk, professor of bone biochemistry at Radboudumc and another of the study’s contributing scientists. “This new technique allowed us to see live interactions at the nanoscale in real time, for example between proteins and calcium. This is critical for studying how tiny particles bind together to form larger complexes, a process that plays a significant role in pathological conditions like kidney stones or vascular calcification.”

Building on old techniques

To build their “super microscope,” the team improved upon an analytical technique called liquid-phase electron microscopy (LP-EM). Unlike traditional electron microscopy, which operates under a vacuum and is not compatible with liquids, LP-EM circumvents the problem of vacuum inside the microscope column, allowing for the study of samples in their natural, hydrated state.

It already has a head start when it comes to observing samples in liquids but its application to soft or biological systems has been limited because the electron beam can damage the delicate biological material. “We use electrons instead of light to achieve nanometric resolution, but the downside is that they generate different reactions when interacting with water that could potentially affect the biological process under observation,” said Macías-Sánchez.

“This is undesirable when you want to observe natural processes […] over extended periods,” added Sommerdijk.

This can be overcome by wrapping the sample in a protective layer, like graphene. “However, the sample preparation process and the localization of the liquid pockets in the grid is time-consuming, wasting precious time in which the reaction has already started,” said Macías-Sánchez. By the time everything is ready — usually at least half an hour later, according to Sommerdijk — the process may already be finished or the desired data not collected.

To overcome this, the team say they took inspiration from the life sciences where a technique called cryogenic correlative light and electron microscopy (cryoCLEM) uses fluorescence microscopy before examining them in detail with an electron microscope.

“We combine light and electron microscopy in a correlative manner,” said Macías-Sánchez. “The sample is encapsulated with a graphene layer, which protects it from the surrounding vacuum, maintaining its liquid structure. The sample is then frozen and observed with fluorescence microscopy to locate areas of interest. It is then transferred to the transmission electron microscope (TEM) and thawed, allowing visualization of the reaction at extremely early timepoints.”

As Ph.D. student Luco Rutten explained, “Biological processes are often very fast, making it challenging to capture their initial stages. This technique allows us to freeze and then re-start biological reactions, enabling us to observe the very start of processes.”

Macías-Sánchez added, “The initiation phases are highly informative, as they determine in many cases the outcome and the physiological response. We know the end point of many biochemical processes, but not the underlying mechanism, so we believe that the development of these types of tools can represent a real leap forward in deciphering these types of mechanisms at the nanoscale.”

Observing calcification in veins

As a proof of concept, the scientists applied this technique to the observation of the process that prevents abnormal calcification in our veins and tissues: the formation of calciprotein particles in cell culture medium, where the protein Fetuin-A binds with excess calcium phosphate.

“In the heart valve on a chip project, we aim to mimic real human heart valves and later introduce calcification,” explained Sommerdijk. By understanding the factors that contribute to valve calcification, we may elucidate the mechanisms and start testing drugs or treatments in this controlled environment, potentially leading to preventative treatments or new therapies for valve-related diseases.”

The team say they benchmarked their new technique against cryogenic TEM, which provides high-resolution “snapshot” images, and dynamic light scattering (DLS), a technique that measures the size and movement of particles in bulk. “These comparisons help confirm that what is seen with the new technique accurately reflects the behavior of natural biological processes,” said Rutten.

While an important step forward, the tech is still a ways out from being able to image live cells, which tend to fall apart under the electron doses of an electron microscope. More research is needed to reduce the electron dose while still maintaining high resolution imaging. “Who knows,” said Macías-Sánchez, “it may be possible in the future. 

For now, Sommerdijk says that they will focus on visualizing processes that occurs in the extracellular space, while also refining imaging techniques to further reduce electron exposure. “The goal is to make this technique more accessible for studying a wide range of biological processes, potentially leading to breakthroughs in how we understand and treat diseases,” he concluded.

Reference: Elena Macías-Sánchez, Nico Sommerdijk, et al., A Cryo-to-Liquid Phase Correlative Light Electron Microscopy Workflow for the Visualization of Biological Processes in Graphene Liquid Cells, Advanced Functional Materials (2024). DOI: 10.1002/adfm.202416938

This article was updated on December 13, 2024 to replace the article’s feature image.