Expansion microscopy technique optimized for viewing meiotic protein structures

By Dr. Stefanie Chen

By testing three different expansion protocols (ProExM, MAP, and U-ExM) and using three types of super-resolution microscopy (dSTORM, SIM, and re-scan confocal microscopy), researchers were able to determine that MAP-SIM worked best for imaging the fine features of the meiosis-specific synaptonemal complex from mouse spermatocytes.

In order to view small molecular structures within a cell using microscopy, images need to be taken below the diffraction limit of light, in a process known as super-resolution microscopy that won the 2014 Nobel Prize in Chemistry. The two main methods used, patterned light illumination and localization-based techniques, require special microscopes and a lot of computational power to produce the super-resolution images. An alternative technique that doesn’t require those resources is being developed.  Expansion microscopy is a sample preparation process in which the intracellular molecules are physically moved farther apart from each other through a gel expansion process, thus increasing the resolution without requiring anything more complex than a standard fluorescent microscope.

Zwettler et al compared several super-resolution and expansion microscopy techniques to view fluorescently-labeled synaptonemal complexes, a special protein structure that holds parental chromosomes together during early meiosis. The synaptonemal complex contains proteins that run longitudinally along the chromosomes, known as the lateral and central elements, and structures perpendicular to those that hold the lateral elements together, known as the transverse filaments. 

First, the researchers tried three previously published expansion protocols, ProExM, MAP, and U-ExM, and found that MAP gave the best results in terms of even fluorescent labeling and expansion across the structure. Then, images of the expanded complexes taken with a type of patterned light super-resolution microscopy called SIM (MAP-SIM) were compared to unexpanded complexes taken with the super-resolution microscopy techniques SIM and STORM (a localization-based technique). MAP-SIM was found to have a similar resolution as STORM, with the added advantages of being able to use three fluorophores for labeling structures and a lower computational burden. 

Using MAP-SIM, the researchers were able to observe the uneven distribution of each of the protein elements within the synaptonemal complex. A novel software that they wrote, called LineProfiler, assisted with the analysis. They also observed fraying of the protein complex along the chromosomes, which occurs as meiosis proceeds.

Developing an established protocol for preparing and viewing molecular structures within the cell opens many doors for investigation of subcellular processes. However, the current protocol (MAP-SIM) may not apply to all cellular structures, as a previous study showed that centrioles only expanded evenly with U-ExM and not MAP. Future research is likely to improve the technique to be more reliable across cellular components, thus lowering the barrier for performing super-resolution microscopy.

Citations

Zwettler, F.U., Spindler, MC., Reinhard, S. et al. Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy. Nat Commun 11, 3222 (2020). https://doi.org/10.1038/s41467-020-17017-7