The researchers at Howard Hughes Medical Institutes Janelia Farm Research Campus, the National Institutes of Health, and Florida State University have developed and applied a new light microscopy technique that can view protein arrangement in cell structures. This said new technique that will allow them to determine the arrangement of proteins that make up the individual organelles, or structures, within a microscopic cell.Eric Betzig, Ph.D., and Harald Hess, Ph.D. while working as independent inventors and later as investigators at Janelia Farm conceived the technique. Betzig and Hess built the light microscope. They demonstrated the method at the NIH, while working with some researchers in the Cell Biology and Metabolism Branch of the National Institute of Child Health and Human Development. Also working on the project was some researchers of the National High Magnetic Field Laboratory at Florida State University.This new light microscope technique is believed to be a major advance that will allow us to understand the fundamental organization of the key structures within a cell. The researchers have learned from this new microscopy technique will provide a broad foundation for understanding the complexity of how proteins, the building blocks of cells, interact in health and disease.The new technique is known as photoactivated localization microscopy or PALM. The technique relies on the effort of some researchers to develop a new class of molecules, called photoactivated fluorescent proteins. These emit green or yellow light when exposed to a laser, but only after being activated by brief exposure to violet light. The cell itself is coaxed to produce these molecules. They are then bound to specific proteins of interest, thereby optically marking the molecular constituents of specific cellular structures.In a conventional optical microscope, objects less than about 200 nanometers apart cannot be distinguished from one another. The trick of the new technique is to control the violet light to activate only a few molecules at a time.This is done so that they are statistically likely to be well separated. Even though each fluorescing molecule still appears as an approximately 200 nanometer diameter spot, the center of the spot, and hence the location of the molecule, can be determined to within 2 to 25 nanometers, depending on its brightness according to the researchers.It is important to activate only a few fluorescent proteins at a time, or else you would only see one bright blur of light, without being able to distinguish the individual position of the protein. By repeating this process many thousands of times, a computer image is eventually created. The image shows the positions of all the molecules, often with near molecular precision. Currently, the main tool researchers use to produce high resolution images of the structures within a cell is an electron microscope. Although electron microscopes produce a detailed image of very small structures, they cannot provide an image of the proteins that make up those structures.

With the new technique discussed in this aticle, the researchers were able to study several cellular subsystems. These include the cells mitochondria, the structures within a cell that provide energy for the cells activities. The researchers were able to visualize the distribution of the proteins involved in the assembly and budding of the AIDS virus from a host cell.

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http://www.nih.gov/news/pr/aug2006/nichd-10.htm