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Science Highlight: October 10, 2014

Nobel Prize Winners in Chemistry Push the Limits of Optical Microscopes

NIBIB scientists worked with Betzig on visualizing proteins, DNA molecules in living cells

The Nobel Prize in Chemistry has been awarded to three researchers for bypassing a law of physics that was thought to limit the size of structures that could be viewed by an optical microscope.

Optical microscopes allow scientists to visualize living cells, bacteria, yeast, and even large organelles, such as mitochondria, in real-time. Yet, molecules such as proteins or individual strands of DNA have remained elusive due to their smaller size. While methods such as electron microscopy can reveal individual molecules, they require the sample to be prepared in such a way that eventually kills it.

The ability to visualize molecules interacting in real-time within a cell is of tremendous value to the biomedical research community. For example, it can help scientists understand how connections between nerve cells in the brain are made; allow individual proteins in fertilized eggs to be followed as they divide into embryos, or enable tracking of proteins involved in Parkinson’s, Alzheimer’s and Huntington’s diseases as they aggregate.

Prior to the work of the three awardees, it was believed that the size of a structure that can be viewed with an optical microscope is limited by the wavelength of visible light. In other words, nothing smaller than half the wavelength of visible light, or one-fifth of a micrometer, could be seen. For reference, the average diameter of a human cell is 50 micrometers, whereas the diameter of double-stranded DNA is two nanometers, 25,000 times smaller.

The three awardees, Eric Betzig of the Janelia Research Campus, Howard Hughes Medical Institute (HHMI), Stefan W. Hell of the Max Planck Institute for Biophysical Chemistry, and William E. Moerner of Stanford University, each independently developed methods that have contributed to scientists’ ability to circumvent this physical limitation.

Hell’s contributions involved the development of a method called stimulated emission depletion (STED) microscopy in which two laser beams are used; one laser makes fluorescent molecules glow and another cancels out all the fluorescence except for a nano-sized portion. Scanning over a sample, nanometer by nanometer, yields a clear image of previously invisible structures.

Betzig and Moerner, working separately, laid the foundation for the second method called single-molecule microscopy. This method involves the ability to turn fluorescent molecules on and off, like a lamp. Scientists image an area within a sample multiple times, allowing only a few interspersed molecules to glow each time. By superimposing many of these images, a composite image of a cell can be created in which individual molecules can be seen.

Revealing Early Development of Life
The first working model of Betzig’s pioneering microscope was built in a lab at the NIH run by Jennifer Lippincot-Schwartz. At the time, Betzig was between jobs, and though he had the theoretical basis for his microscope, he had neither the green fluorescent proteins needed for the experiment nor access to a laboratory in which to build it.

George Patterson, Lab Chief of the Section on Biophotonics in NIBIB’s Intramural Research Program (IRP), worked with Betzig during this critical time and played an essential role in creating the highly-specialized photoactivatible fluorescent probes needed to enable single-molecule microscopy.

“After developing a photoactivatable version of the green fluorescent protein during my postdoctoral fellowship with Jennifer Lippincott-Schwartz, I knew it would be a useful addition to the cell biology imaging toolbox. However, when Eric described his idea over lunch after one of his seminars, the full potential of photoactivatable probes became clear. He had just described something revolutionary in the field,” said Patterson.

Hari Shroff, Chief of NIBIB’s Section on High Resolution Optical Imaging, also worked with Betzig, as a postdoctoral student in his lab at HHMI. “I learned a great deal from Eric; and his work ethic was inspiring,” said Shroff. Shroff has since worked to extend the imaging methods he learned in Betzig’s lab, adapting them for 3D imaging in thicker samples.

Moerner also has connections to NIBIB, having served on the Board of Scientific Counselors for NIBIB’s Intramural Research Program for four years.

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