|New Light Microscope Can View Protein Arrangement in Cell
Researchers at Howard Hughes Medical Institute’s Janelia Farm Research Campus,
the National Institutes of Health, and Florida State University have developed
and applied a new light microscopy technique that will allow them to determine
the arrangement of proteins that make up the individual organelles, or structures,
within a cell.
The microscope and the technology that make it possible are described in an
article appearing on-line in the August 10 issue of Science Express.
The technique was conceived by Eric Betzig, Ph.D., and Harald Hess, Ph.D. while
working as independent inventors and later as investigators at Janelia Farm,
which subsequently supported their effort on the project. Funding for the project
was also provided by the NIH. Drs. Betzig and Hess built the microscope and demonstrated
the method at the NIH, while working with Jennifer Lippincott-Schwartz, Ph.D.
and her colleagues in the Cell Biology and Metabolism Branch of the National
Institute of Child Health and Human Development. Also working on the project
was Michael Davidson of the National High Magnetic Field Laboratory at Florida
“This is a major advance that will allow us to understand the fundamental organization
of the key structures within a cell,” said Elias A. Zerhouni, M.D., Director
of the NIH. “What researchers learn from the 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 (PALM).
It relies on the earlier pioneering effort of Dr. Lippincott-Schwartz and NIH
Staff Scientist George Patterson, Ph.D. to develop a new class of molecules,
called photoactivated fluorescent proteins, which 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, which 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, 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.
“It’s important to activate only a few fluorescent proteins at a time, or else
you’d only see one bright blur of light, without being able to distinguish the
individual position of the protein,” Dr. Lippincott-Schwartz said.
Repeating this process many thousands of times, a computer image is eventually
created in which the positions of all the molecules are determined, 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, the researchers were able to study several cellular
subsystems, including the mitochondria, the structures within a cell that provide
energy for the cell’s 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.
The images depict a membrane
protein in a cellular organelle known as a lysosome. The image on the
right shows a convention fluorescent image of a portion of the lyososome,
whereas the image on the left shows the corresponding PALM image in the
Images generated by both conventional microscopy and the new PALM microscopy
appear at http://www.nichd.nih.gov/new/releases/caption_palmvsconventional.cfm.
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child, and family health; reproductive biology and population issues; and medical
rehabilitation. For more information, visit the Institute’s Web site at http://www.nichd.nih.gov/.
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Agency — includes 27 Institutes and Centers and is a component of
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agency for conducting and supporting basic, clinical and translational medical
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