By KRISTIN WEIDENBACH

Since the early part of the 17th century, scientists have used microscopes to spy on critters and cells that cannot be seen with the naked eye. Light microscopes allow researchers to look at bacteria or single-cell algae, the cells in a smear of blood or those clustered at the tip of a plant root. But when scientists wish to see the tiny molecules and structures inside those cells they turn to the electron microscope, which uses a beam of electrons instead of light as its source of illumination.

A group of Stanford scientists has now found a way to extract more information than ever before from electron microscope images and in so doing has begun to unravel a long-standing mystery of neuroscience.

U.J. McMahan, PhD, professor of neurobiology, and his research group -- consisting of research associates David Ress, PhD, and Robert Marshall, PhD; graduate student Mark Harlow; and Arne Stoschek, PhD, a former postdoctoral fellow in McMahan's lab -- are using the new technology of electron microscope tomography, which has been developing over the last decade.

"The advantage of EMT over conventional electron microscopy is that it provides three-dimensional information," said McMahan. "Conventional electron microscopy provides only two-dimensional images of tissue sections so there is no information as to where a structure is positioned within the depth of a section. EMT uses a series of 2-D images of a section made at different tilt angles to computationally reconstruct the three-dimensional volume."

The researchers are using EMT to study active zone material -- protein clumps that gather near the gap between the end of one neuron and the beginning of another. Scientists have known of this material for 50 years but have not known what it does. "The proteins are very small and very densely packed together," he said. "Even with the electron microscope and the thinnest sections you can possibly cut, it just looks like a very dark blob."

Scientists have been intrigued by this mysterious material because of its esteemed location at the very tip of the transmitting end of a neuron. When an electrical signal travelling along a nerve cell reaches the tip, it must cross a gap, called the synapse, before continuing along the next neuron. The signal crosses the synapse via the release of chemicals called neurotransmitters, which are contained in small vesicles until they are needed. When an electrical signal reaches the end of the transmitting neuron, or pre-synaptic cell, calcium floods in through special channels and the vesicles release their chemicals, which transmit the signal across the synapse to the receiving nerve cell. The active zone material is found among the vesicles clustered at the end of the pre-synaptic nerve cell.

Using conventional electron microscopy the active zone material appears as small, flat blobs. Using the new technique the shapeless blobs materialize into structures with "ribs," "beams" and "pegs."

"EMT allows us to take a flat image and reconstitute it back into its 3D state," said Harlow, the graduate student on the project and lead author of a paper published in the Jan. 25 issue of Nature describing the results of the team's study. "It allows us to go in and look at clusters of proteins and how they're organized. There haven't been the techniques to look at large groupings of cell structures in natural tissue at a really fine level of detail."

According to McMahan, three-dimensional reconstructions from EMT data often have been hampered by poor clarity of the resulting images. At such high magnification every speck or blob in the cell is accentuated, sometimes combining to obscure what the scientist is striving to see. "People have made 3-D reconstructions but they're very 'noisy,' so the information you got out was extremely limited," he said. The computer program he and his colleagues developed enables the user to home in on particular cell structures and filter out this background interference.

The 3-D images revealed that the active zone material has three components the team has named ribs, beams and pegs. These components interact to form a type of scaffolding that hooks into the surrounding vesicles and calcium channels in the pre-synaptic nerve cell to hold them in position. The ribs are connected to docked vesicles and the pegs appear to be connected to calcium channels. To date, the researchers have analyzed only the thin layer of active zone material closest to the outside of the cell.

McMahan said his group plans to inspect the rest of the active zone material and its association with neuronal cytoskeleton.