When it comes to fluorescence microscopy, a team of investigators has shown that you can see more with less.Scientists at the Janelia Farm Research Campus of Howard Hughes Medical Institute (HHMI) in Ashburn, Va., at the National Institute of Child Health and Human Development in Bethesda, Md., at Florida State University in Tallahassee and at NuQuest Research LLC in La Jolla, Calif., imaged fluorescent proteins within cells with ~2- to 25-nm resolution. That is far below the classical resolution limit of half a wavelength, or a few hundred nanometers in the visible.“Resolution is a key advantage compared with optical microscopy. The key advantages compared with electron microscopy are related to contrast,” said Eric Betzig of HHMI. He invented the method, dubbed photoactivated localization microscopy, along with Harald F. Hess of NuQuest.Both benefits, Betzig explained, result from the use of photoactivated fluorescent proteins. The contrast enhancements occur because the proteins are expressed by the cell itself after genetic modification. This internal production eliminates the potentially perturbing sample-preparation steps involved in electron microscopy and is more discriminatingThe resolution is achieved by serial activation of the proteins’ fluorescence. In their proof-of-concept research, the scientists first fired a 405-nm pulse from a Coherent Inc. diode laser with intensity and duration chosen to activate sparse fields of individually resolvable molecules. They imaged each protein in turn using a beam from a 561-nm solid-state laser made by Lasos Lasertechnik GmbH of Jena, Germany, until the proteins photobleached. They captured the resulting images using a cooled, electron-multiplying CCD camera from Andor Technology of Belfast, UK, fitting the observed profile to the ideal 200-nm-wide point spread function.Because the investigators had many photons for the fit, they could pinpoint the proteins’ locations to within a few nanometers. Importantly, the individual images did not overlap. “Only in this way is it possible to determine the center of each spot,” Betzig said.In demonstrations, they imaged specific target proteins in lysosomes and mitochondria as well as various targets in fixed, whole cells, repeating the process of activation and photobleaching as many as 100,000 times.A conventional optical image of lysosomes within a cell is compared with a subdiffraction-limit image (upper left) — acquired using a technique called photoactivated localization microscopy — taken from the smaller boxed region at right.A drawback to the technique is the imaging time, which runs between two and 12 hours. In addition, the fluorescent proteins do not all produce the same number of photons, so the localization precision varies.Betzig noted that the latter issue might be helped by increasing the photon flow through various techniques. The imaging time might be reduced to five minutes or so by increasing the laser power and the number of photoactivated molecules in each frame. Finally, he noted that the technique’s future as a research tool might be helped by its simplicity. “It might not require much modification of existing commercial systems to add this capability,” he said.Sciencexpress, Aug. 10, 2006, doi:10.1126/science.1127344.