Sep 11, 2015 -- STOWERS INSTITUTE FOR MEDICAL RESEARCH -- KANSAS CITY, MO -- Cellular mitosis depends in part on small organelles that extend spindles to pull apart chromosome pairs. Before they can perform this and other essential tasks, these tiny cylindrical structures -- known as centrioles in animals and spindle pole bodies (SPBs) in yeast -- must themselves duplicate.

However, much about this nanoscale process has remained veiled by the limits of current microscopy. Optical approaches cannot resolve objects below certain wavelength limits, while non-optical approaches like electron microscopy (EM) can only study nonliving cells.

Now, a team of researchers from the Stowers Institute for Medical Research and the University of Colorado Boulder has devised a novel optical technique -- a combination of structured illumination microscopy (SIM) and single-particle averaging (SPA) -- to resolve individual components of SPB duplication in living yeast cells. In the process, they have uncovered surprising facts in what many once considered well-trodden ground. More than that, they have opened up new possibilities in the field of cellular imaging.
"The use of SIM to study SPB structure completely changes the types of questions we can ask and answer, since sample size is no longer limiting and it is likely that SIM will work in living cells," says Sue Jaspersen, Ph.D., an associate investigator at the Institute who led the investigation.

The study will be published in the online journal eLife on September 15, 2015.

For years, EM, which uses a beam of electrons to achieve molecular and even atomic resolutions, has been the go-to technique for studying SPBs, which at less than 200 nanometers (nm) in size fall below the wavelength limit of what is observable using visible light. However, EM carries with it significant limits, including the fact that it does not work on living cells.

The research team turned to SIM as an optical alternative. SIM uses a laser-generated field of horizontal lines to project an interference pattern onto a sample. According to Jay Unruh, Ph.D., a Stowers research advisor and co-author, analyzing these patterns enables researchers to effectively double their resolution.

"Basically, finely structured objects interfere with the line pattern in a way that makes a new pattern, which is larger in size."

For all of its advantages, SIM still involves sifting through a great deal of noise. To deal with this problem, and to better localize the subjects under study -- which can assume various shapes and positions -- the team turned to SPA. In this technique, researchers align a large number of images along reference points in three-dimensional space and then average them into a single, characteristic image. The result is a sharper, more reliable picture of what is going on within the cell.

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