Having a greater understanding of cells’ ultrastructure is on the wish list for an array of researchers. While advances in technology over the years have provided investigators with unprecedented views into biological processes and molecular and cellular structures, it has been met with some limitations. Now, a team of scientists at Daegu Gyeongbuk Institute of Technology (DGIST) in South Korea has recently developed a new methodology for nanoimaging, which is widely used to structurally characterize subcellular components and cellular molecules such as cholesterol and fatty acids. Using a molecule called graphene, a single layer of carbon atoms arranged in a two-dimensional honeycomb lattice, the researchers were able to coat cells and keep them alive and wet for much-improved imaging.

“Most advanced nanoimaging techniques use accelerated electron or ion beams in ultra-high-vacuum environments,” explained senior study investigator Dae Won Moon, PhD, professor at DGIST. “To introduce cells into such an environment, one must chemically fix and physically freeze or dry them. But such processes deteriorate the cells’ original molecular composition and distribution.”

Findings from the new study—published recently in Nature Methods through an article titled “Mass spectrometry imaging of untreated wet cell membranes in solution using single-layer graphene“—could help researchers gain a greater understanding of mechanisms underlying diseases such as cancer, Alzheimer’s, and others.

“We wanted to apply advanced nanoimaging techniques in ultra-high-vacuum environments to living cells in solution without any chemical and physical treatment, not even fluorescence staining, to obtain intrinsic biomolecular information that is impossible to obtain using conventional bioimaging techniques,” remarked lead study investigator Heejin Lim, PhD, a key member of the research team at DGIST.

The researchers’ technique involves placing wet cells on a collagen-coated wet substrate with microholes, which in turn is on top of a cell culture medium reservoir. The cells are then covered with a single layer of graphene. It is the graphene that is expected to protect the cells from desiccation and cell membranes from degradation.

Through optical microscopy, the scientists confirmed that, when prepared this way, the cells remain viable and alive up to ten minutes after placing in an ultra-high-vacuum environment. The scientists also performed nanoimaging, specifically, secondary ion mass spectrometry imaging, in this environment for up to 30 mins. The images they captured within the first ten minutes paint a highly detailed (submicrometer) picture of the true intrinsic distribution of lipids in their native states in the cell membranes; for this duration, the membranes underwent no significant distortion.

“We report a means by which atomic and molecular secondary ions, including cholesterol and fatty acids, can be sputtered through single-layer graphene to enable secondary ion mass spectrometry (SIMS) imaging of untreated wet cell membranes in solution at subcellular spatial resolution,” the authors wrote. “We can observe the intrinsic molecular distribution of lipids, such as cholesterol, phosphoethanolamine, and various fatty acids, in untreated wet cell membranes without any labeling. We show that graphene-covered cells prepared on a wet substrate with a cell culture medium reservoir are alive and that their cellular membranes do not disintegrate during SIMS imaging in an ultra-high-vacuum environment.”

With this method, a cascade of ion beam collisions at a point on the graphene film can create a big enough hole for some of the lipid particles to escape. But interestingly, while this degradation to the cell membrane does occur, it is not significant within the ten-minute window, and there is no solution leakage. Further, the graphene molecules react with water molecules to self-repair. So, overall, this is a great way to learn about cell membrane molecules in their native state in high resolution.

“I imagine that our innovative technique can be widely used by many biomedical imaging laboratories for more reliable bioanalyses of cells and eventually for overcoming complex diseases,” Moon concluded.

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