Researchers at the Daegu Gyeongbuk Institute of Technology (DGIST) have introduced a method for analyzing subcellular structures using graphene. The work holds implications for the research of biological processes such as the mechanisms of certain diseases. According to the researchers, the impetus for the work stems from a desire to step away from conventional imaging techniques that are unable to deliver insight into a sample’s structures. Professor Dae Won Moon (sitting) and Dr. Heejin Lim (standing) in their lab at the Daegu Gyeongbuk Institute of Technology (DGIST). Courtesy of DGIST. “Most advanced nanoimaging techniques use accelerated electron or ion beams in ultrahigh vacuum environments. To introduce cells into such an environment, one must chemically fix and physically freeze or dry them,” said Dae Won Moon, a professor in DGIST’s department of new biology. “But such processes deteriorate the cells’ original molecular composition and distribution.” “We wanted to apply advanced nanoimaging techniques in ultrahigh 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,” research team member Heejin Lim said. The approach involved placing wet cells on a collagen-coated wet substrate with microholes, itself positioned on top of a cell culture medium reservoir. The cells were then covered with a single layer of graphene. The researchers theorized that the graphene would protect the cells from dessication and would protect the cell membranes from degradation. Using optical microscopy, the researchers confirmed that the cells remained living, and, for their intents and purposes, viable for up to 10 minutes in an ultrahigh vacuum environment following this method of preparation. They also performed nanoimaging, specifically secondary ion mass spectrometry imaging in this environment for up to 30 minutes. Images obtained in the first 10 minutes displayed a submicrometer-resolution image of the true intrinsic distribution of lipids in their native states within the cell membranes. The membranes showed no significant distortion during this time. The researchers did notice, however, that a cascade of ion beam collisions at a point on the graphene film can create a hole big enough for some of the lipid particles to escape. That effect was not significant within the 10-minute window, and there was no solution leakage. Further, interactions between the graphene molecules and the water molecules allowed the graphene to self-repair. “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 said. The research was published in Nature Methods (www.doi.org/10.1038/s41592-020-01055-6).