A new superresolution microscopy technique is answering longheld questions about how and why a cell’s defenses fail against some invaders, such as plague, while successfully fending off others, such as E. coli. The approach is revealing never-before-seen details of the cell membrane, which could open doors to novel diagnostic, prevention and treatment techniques. “We’re trying to do molecular biology with a microscope, but in order to do that, we must be able to look at things on a molecular scale,” said Jesse Aaron of Sandia National Laboratories, where the technique was developed. The difference between what was previously seen on the cell surface (left image) is dramatically different from what researchers at Sandia National Laboratories have been able to image (right). Orange areas correspond to the bacterial lipopolysaccharide (LPS), derived from E. coli, and the green areas correspond to the cell’s TLR4 receptors. (Image: Jeri Timlin, Jesse Aaron and Bryan Carson) The cell membrane is a bustling hub of activity on a minuscule scale. While providing structure and housing for the cell’s interior, the membrane regulates movement of materials in and out of the cell, controls adhesion to other objects and coordinates the cell’s communications and subsequent actions through signaling. Receptor proteins on the surface of immune cells, known as toll-like receptors (TLRs), are designed to recognize antigens. The TLR4 member of this receptor family responds to certain types of bacteria by detecting lipopolysaccharides (LPS) present on their surface. TLR4 proteins then alert the cell and activate an immune response. Using imaging techniques they developed, Aaron and Sandia colleagues Jeri Timlin and Bryan Carson discovered that TLR4 proteins cluster in the membrane when confronted with LPS derived from E. coli, which increases cell signaling and response. Interestingly, LPS derived from the bacteria that cause plague, Yersinia pestis, do not cause the same effects. This could explain why some pathogens can thwart the human immune system. Jesse Aaron, left, Jeri Timlin, and Bryan Carson in their laboratory working with new imaging techniques to view cell-level activity with unprecedented detail. (Image: Randy Montoya) The plague studies marked the first time such small events have been imaged and compared, the Sandia researchers say. Previously, even the most sophisticated optical microscopes could not image the cell surface with enough spatial resolution to see the earliest binding events. The main problem is that standard optical microscopes cannot exceed the diffraction barrier, which limits what can be resolved using visible light. “With more traditional visualization methods, you can’t see the level of detail you need. It’s important to look at not only what’s present, but also when and where it’s present in the cell,” Timlin said. The technique used by Timlin and Aaron builds on the superresolution capability of stochastic optical reconstruction microscopy, or STORM, but goes another step by adding dual-color capabilities. The combination enabled the Sandia team to get a more complete picture by simultaneously imaging LPS and TLR4 receptors on the membrane. “Current light microscopy capabilities are akin to looking out the window of an airplane and seeing the irrigation circles. You know that plants are there, but you can’t tell what kinds of plants they are or what shape the leaves are,” Carson said. “But with this technology, it’s like zooming in and seeing the leaves and the structure of the plants. That buys you a lot in terms of understanding what’s happening within a cell and, specifically, how the proteins involved interact.” For more information, visit: www.sandia.gov