Researchers have refined a micro-scope that allows them to image features far smaller than the diffraction limit of the light used and examine how a protein behaves inside a living cell.
At May’s Conference on Lasers and Electro-Optics (CLEO), post-doctoral researcher Julie Biteen described how she and others in W.E. Moerner’s group at Stanford University sequentially turned on different fluorescent label subsets, allowing them to create images of proteins within live cells with sub-40-nm resolution (Talk CFT2, “Superresolution Imaging in Live Bacterial Cells by Single-Molecule Active-Control Microscopy”).
Fluorescence microscopy has been useful to biologists studying cells and sub-cellular structures. However, the diffraction limit for visible light limits the resolution of normal microscopes. Moving to shorter wavelengths would also mean increasing the energy of the photons; this damages living cells as one approaches the edge of ionizing radiation.
But there are ways around the diffraction limit. If one uses low concentrations of small emitters that are far enough apart when they fluoresce, the position of the nanoscale emitters can be determined more precisely than the standard diffraction limit.
When a higher concentration is needed, the researchers suggest acquiring many measurements from low-concentration subsets of the labels in order to build up an image. This technique, called photoactivatable light microscopy (PALM), was reported by Eric Betzig and his colleagues in 2006 (Science 313, 1642).
Biteen and coworkers wanted to look inside live cells at superstructures of an actin-like protein called MreB, which, in bacteria such as E. coli and Caulobacter crescentus, is critical for determining cell shape and division. “Bulk low-resolution imaging experiments indicated that this protein forms a superstructure that is dynamic over the course of the cell cycle, suggesting helices in stalked cells and midplane rings in pre-divisional cells,” said Biteen. Thus far, nobody had directly observed the protein organization.
The group used single-molecule active-control microscopy (SMACM), a variation on PALM, to obtain superresolution images (which resolve features smaller than 40 nm) of this polymerized protein chain. The images showed previously unseen structures in the live bacteria during different stages of its life (see above). The top two images took 2 to 3 min. to acquire and the bottom two took about 15 min.
The scientists labeled the MreB with a fluorescent protein called EYFP. Unlike the photoactivatable fluorescent molecules traditionally used in superresolution experiments, which can be activated only once and eventually stop emitting, EYFP can be reactivated. If the EYFP-MreB structures worked the same way in living cells, researchers could determine the location of a single emitter multiple times, thus improving the effective labeling density. And that’s just what they did, providing evidence of photoinduced EYFP reactivation in live cells.