"Small deviations that can be due to noise are eliminated, and we can localize proteins with high confidence, often with even greater precision than via SIM alone," says Jaspersen. "We estimate the precision to be in the 10-30 nm resolution range."

The SPA-SIM technique made up part of a two-color structured illumination microscopy approach that used endogenously expressed fluorescent protein derivatives. Through genetic techniques, the researchers fused the proteins in question to two standard fluorescent proteins, which would show up when the yeast naturally expressed the gene.


According to Unruh, this study represents the first combined use of SPA with SIM, and one of the first dual-color super-resolution SPA papers. Moreover, where other studies concentrated on a few protein pairs, the current study "characterized a significant portion of a large, many-protein complex."

"The big-picture take away is that even structures that are considered by many to be 'solved' -- we know what they look like by EM, we know all the parts, we know many of the physical interactions between those parts -- have remarkable surprises when one is finally able to study their formation in cells."

Among those surprises: SPB duplication, once thought to occur in the G1 period of interphase, now appears to begin near the end of mitosis. Moreover, the structures by which SPBs attach to the nuclear envelope do not form at the end of duplication, as once thought, but rather during the duplication process itself.

The study also reported a number of never-before-seen structural features, including "the structure and timing of half-bridge elongation, the composition of the satellite and the formation of the membrane pore."

But that's just the beginning. According to Jaspersen, the SPA-SIM technique is applicable to a wide variety of subjects beyond SPB structure.

"This method can be applied to any regular intracellular structure if there is some known reference protein that can be used."

Other Stowers authors are Shannon Burns, Zulin Yu, Ph.D., Sarah Smith, Ph.D., and Brian Slaughter, Ph.D. Additional contributors include Jennifer Avena, Ph.D., and Mark Winey, Ph.D., at the University of Colorado, Boulder.

The study was funded by the Stowers Institute and the National Institutes of Health (Mark Winey, P01 GM105537; Jennifer Avena, 5 T32 GM007135; Jennifer Avena, Mark Winey, R01 GM51312). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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