Researchers from Radboud University Medical Center and Biointerface Laboratory RWTH Aachen University Hospital have used graphene to develop a new technique that allows them to image biological processes as they occur, with enough detail to see protein complexes move. They have demonstrated the method by showing, for the first time, how calcium deposits into a form that may lead to calcification of the arteries and aortic valve.
Schematic overview of the cryo-to-liquid-CLEM workflow. Image from: Advanced Functional Materials
The team explained that liquid phase electron microscopy (LP-EM) has emerged as a powerful technique for in situ observation of material formation in liquid. The use of graphene as window material provides, according to the scientists, new opportunities to image biological processes because of graphene's molecular thickness and electron scavenger capabilities. However, in most cases the process of interest is initiated when the graphene liquid cells (GLCs) are sealed, meaning that the process cannot be imaged at early timepoints. So, they developed a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses the delay time between graphene encapsulation and the start of the imaging, while combining the advantages of fluorescence and electron microscopy.
“If you want to see protein complexes in such fine detail, you need an electron microscope,” says Nico Sommerdijk, professor of bone biochemistry at Radboud University Medical Center in the Netherlands. “But the electron beam used can damage the biological material and the surrounding fluid, which is undesirable when you want to observe natural processes in the material over extended periods.”
Researchers have been able to reduce the radiation damage associated with this technique called liquid-phase electron microscopy, by applying a protective layer of graphene over the sample, however: “As soon as you apply it [the graphene], the biological process you want to capture starts immediately,” Sommerdijk explained. “And then you have to quickly reach the microscope, locate the right spot in the tissue, and set up the microscope. This process takes at least half an hour, and sometimes the process is already over by then.”
So Sommerdijk and the team came up with a new method to overcome these limitations, that involves adding a non-reactive fluorescent dye when preparing the sample, before applying a layer of graphene around it. Then, they plunge the sample into liquid ethane to immediately freeze it and stop all biological processes in their tracks. Thanks to the fluorescent dye, they can use a normal microscope to identify the specific area in the sample they want to look at, avoiding any further damage associated with using an electron microscope.
Then the material is placed in the electron microscope and allowed to thaw. This reactivates the biological processes, which can then be visualized without delay.
The researchers tested their new method by capturing a biological process that prevents calcification from occurring in arteries.
Study first author, Luco Rutten, a PhD candidate at Radboud University Medical Center in the Netherlands, said: “If there’s too much calcium phosphate in the blood, a particular protein in the body can bind to it, preventing it from precipitating. The kidneys then clear it out. Under the microscope, we see that these proteins form tiny spheres with calcium phosphate, which can still be broken down. But these spheres can also grow larger, causing calcium phosphate to turn into calcified deposits, which can no longer be broken down.”
The researchers think this may contribute to calcification in the body.
“We still don’t fully understand what exactly happens with this type of calcification, which is why there are no medications yet,” added Sommerdijk, who now plans to study arterial calcification further with the new microscope technique.