A team of researchers from the Stowers Institute for Medical Research and the University of Colorado Boulder has designed a novel optical technique which uses a combination of structured illumination microscopy (SIM) and single-particle averaging (SPA). It is devised to resolve individual components involved in duplication of spindle pole bodies in living yeast cells. This innovative technique opens new possibilities in the field of cellular imaging and uncovers surprising facts in what many once considered well-trodden ground.
The online journal eLife published the study on September 15, 2015.
The process of cellular mitosis depends in part on small organelles like centrioles in animals and spindle pole bodies (SPBs) in yeast. These organelles extend spindles to pull apart the pairs of chromosome. However, they are supposed to duplicate themselves before performing such crucial tasks. For most of the time, this nanoscale process has remained veiled by the limits of current microscopy. Optical approaches cannot resolve objects below certain wavelength limits. Moreover, non-optical approaches like electron microscopy (EM) can only study nonliving cells.
According to the researchers, using SIM to study SPB structure completely changed the types of questions one can ask and answer. The sample size is no longer limiting and it is likely that SIM will work in living cells as well.
EM uses a beam of electrons to achieve molecular and even atomic resolutions. It has been serving as the go-to technique for studying SPBs for years. SPBs are less than 200 nanometers (nm) in size and fall below the wavelength limit that is observable using visible light. There are certain limitations residing EM including the fact that it does not work on living cells.
SIM as an optical alternative
SIM served as an optical alternative for the research team. It uses a field of horizontal lines, generated by the laser, to project an interference pattern onto a sample. Analyzing these patterns caused the researchers to effectively double their resolution. The laser-generated line pattern interferes with the finely structured one in such a way that makes a new large-sized pattern. On the other hand, SIM involves going through a great deal of noise. In order to deal with this problem, and to better localize the subjects under examination the Stowers team turned to SPA.
This technique involves aligning a large number of images along with reference points in a three-dimensional space. Afterward, the images are regulated into a single, characteristic image. The resulting image provides a sharper and reliable picture of what is going on inside the cell.
SPA is estimated to eliminate small deviations that can be due to noise. Plus it helps to localize proteins with high confidence and greater precision than via SIM alone. The researchers estimate the precision to be in the range of 10–30 nm resolution.
This respective study represents the first combined use of SPA with SIM and one of the first dual-color super-resolution SPA papers. Previous studies are concentrated on a few protein pairs whereas; the current study characterizes a notable portion of a large, many-protein complex. It reveals the potential features of the cellular structures. For instance, SPB duplication was thought to occur in the G1 period of interphase. However, it now appears to begin near the end of mitosis. Furthermore, it was thought that the structures by which SPBs attach to the nuclear envelope do not form at the end of duplication but rather during the duplication process itself.
The study also reports a number of structural features that were never seen before. This includes,
- Structure and timing of half-bridge elongation
- Composition of the satellite
- Formation of the membrane pore
The research team comments that the SPA-SIM technique is applicable to a wide variety of subjects beyond the SPB structure. This study is just the beginning. This method can be applied to any regular intracellular structure. If only some known reference protein is present and known.