A “LITTLE” Microscopy News

By: Kerry Connell

Nobel Prize winner Eric Betzig is at it again. In the 2014 edition of Issue 4 of Lab Reporter, we reported that Dr. Betzig, William Moerner and Stefan Hell had won the Nobel Prize in chemistry for using fluorescence to expand the resolution of optical microscopes. That advancement permitted scientists to observe molecules within living cells in real time and broke the longstanding rule set forth in 1873 by Edward Abbe, who determined that the “diffraction limit” of light makes it impossible to see separate molecules that are closer together than 200 nanometers.

The 2014 research findings took fluorescence microscopy to a new level, but a scientist never stops asking questions. In his current research, Dr. Betzig has been attempting to provide even more precise tools to help researchers in molecular, cellular and neurobiology see even farther into the living organism. He has achieved at least one of his goals: thanks to his work with structured illumination microscopy (SIM), the resolution level now stands at 45 to 84 nanometers wide. In this game, smaller is better.

SIM: The Little Picture

The August 28, 2015, issue of Science includes the latest on Dr. Betzig’s efforts. “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics” elucidates the methods by which he and his research team sought to extend the range previously established by the Nobel winning work with super resolution (SR) fluorescence microscopy.

SR fluorescence microscopy does allow for intricate examination of the organism, but the intense light that the process entails can profoundly disturb the physiology in question. SIM is faster and requires far less light than other SR approaches — but, again, the method’s resolution is “usually limited to only a twofold gain beyond conventional optical microscopes, or ~100 nm with visible light.”

The scientists first employed an ultrahigh numerical aperture lens and total internal reflection fluorescence to achieve 84nm resolution. Next, they used a newly developed fluorescent protein —which is reversibly photoswitchable — to reach 45 to 62nm resolution. Notably, both approaches were workable at sub-second acquisition speeds over hundreds (in the first case) or dozens (in the second case) of time points. Experimental comparisons showed that the resolution of these methods is as good as or better than other SR approaches — but it can be attained at far higher speeds.

Get Ready for Big Changes in Biological Research

How can this possibly work? The scientists developed a theoretical model that explains the breakthrough: these methods transmit the information encoded in spatial frequencies beyond the diffraction limit, with much greater strength than other methods. Therefore, the specimen is required to emit far fewer photons. Fewer photons means less intense light. The ability to watch sub-second interactions among cell structures is the result — a result that opens the door to potentially significant discoveries.

Quick images of living organisms without as much harmful light? That sounds like a big deal to us!

Sidebar Spotlight: Eric Betzig, Ph.D.

Dr. Betzig, of the Janelia Research Campus of the Howard Hughes Medical Institute in Ashburn, Virginia, is always looking for ways to extend the capability of optical microscopy. He and his multidisciplinary team of researchers focus on five areas that they believe need improvement:

Spatial Resolution:

Cells that express fluorescent proteins tell us about the spatial organization of target proteins at the molecular level, but conventional optical microscopy is limited in its resolution by diffraction. A system that can image intracellular proteins with near-molecular resolution could determine the static structural relationship between two or more proteins.

Temporal Resolution:

Studying dynamic processes is more interesting, so the team seeks to extend spatial resolution beyond the diffraction limit without sacrificing temporal resolution. They are working on an optical lattice microscope, composed of a massively parallel array of excitation foci, to be used with new resolution methods.

Labeling Technology:

Higher performance is possible through the synthesis of new optical labels with better photophysical properties. The team hopes to identify and refine probes for application to biological studies in conjunction with new imaging modalities.

Deep Tissue Imaging:

The team is exploring the imaging of intra- and intercellular processes in the brain’s neural circuits at high spatiotemporal resolution using a variety of techniques.

Noninvasive, Datarich Imaging

The team is developing imaging techniques that confine the optical field strictly to the points of interest to allow researchers to create a densely sampled, multidimensional measurement volume covering a broad span of space, time and structural and functional contrast.