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Imaging the synaptonemal complex in 3D-SIM on the DeltaVision OMX

Discover how super resolution imaging of the synaptonemal complex furthers our understanding of its function

DeltaVision OMX SR

Why is the synaptonemal complex a great model for 3D-SIM?

Early in meiosis, homologous chromosomes are held together by a zipper-like structure called the synaptonemal complex (SC). Literature shows that defects in the human SC lead to problems with chromosome segregation that are associated with infertility, recurrent miscarriage, and genetic disorders like Down’s Syndrome (1, 2). Researchers have been studying the SC since 1956, but due to its very small size, they have been unable to resolve structural and spatial details required to understand the relationship between the SC’s structure and function. In the past few years, there have been several important discoveries in this field using super-resolution imaging techniques, like 3D Structured Illumination Microscopy (3D-SIM), which have led to a better understanding of the SC and associated proteins.

The width of the SC makes it an ideal structure to study with structured illumination. 3D-SIM on the DeltaVision OMX can achieve resolution of 120 ± 5 nm in the x and y axes and 340 ± 10nm in the z axis (when imaged with the 488 laser), while the SC varies in size from 100 to 220 nm depending on the model organism.

Highlights from the publication: A mammalian KASH domain protein coupling meiotic chromosomes to the cytoskeleton

Horn, H. et al. A mammalian KASH domain protein coupling meiotic chromosomes to the cytoskeleton J. Cell Biol. 202(7), 1023–39 (2013), doi:10.1083/ jcb.201304004.

Experiment: What does the SC-binding protein KASH5 do?

When Henning Horn started his postdoc in Colin Stewart’s lab at the Institute of Medical Biology (IMB) in Singapore, his first project was to investigate the role of a protein named KASH5 in male meiosis of mice. His colleagues had already shown that KASH5 localized to the tips of the SC but when Henning used a widefield fluorescence or confocal microscope to image spermatocyte spreads from Kash5-null mice, he was unable to resolve the two axial elements (AE) of the SC. He knew that being able to do so would add novel information to their study. Henning showed his images to Graham Wright, the Head of the Microscopy Unit, an IMB core facility, who immediately recognized that the SC was a perfect biological structure to image using 3D-SIM on the DeltaVision OMX super-resolution microscope. With new tools at their disposal, the authors set out to further characterize KASH5. Specifically, they wanted to determine if KASH5 functioned similarly to other previously characterized KASH proteins which tether the chromosomes to the cytoskeleton.

How does the DeltaVision OMX workflow compare to an EM workflow?

Once 3D-SIM was identified as an option, the authors were excited to see if the ability to separate AEs of the SC would allow them to more precisely identify where KASH5 localized in the context of the SC. While the authors could use their existing fluorophores and their standard sample preparation protocol, Henning recalls that some optimization was needed to acquire high-quality DeltaVision OMX images. Specifically, mounting the samples on the coverslips (rather than on the microscope slide) and changing to a soft mountant was required. Once sample preparation was optimized, this opened the door for additional experiments.

Graham noted that the DeltaVision OMX allowed them to obtain high-quality datasets just days after preparing the samples. He notes that it would have taken 3 to 4 weeks to obtain results, had they decided to use electron microscopy (EM) instead of 3D-SIM. Additionally, even though EM can achieve higher resolution than 3D-SIM, it does not produce 3D datasets, thus limiting the spatial information they could gather and eliminating the possibility of chromosome quantification.

Discoveries in 3D-SIM on the DeltaVision OMX

Spermatocyte spreads

Fig 1. Spermatocyte spreads, labeled with anti-KASH5 in green and anti-SCP3 in red, imaged by widefield deconvolution microscopy. SCP3 is visualized as a single strand per chromosome. KASH5 localizes as a large foci to the tips of SCP3-positive SCs. B. Structured illumination microscopy of the same cells imaged in A. The two AEs of the SC are clearly resolved with SCP3 staining and KASH5 localizes as rings at the tips of the SCP3 axial strands. C. Enlargement of A. D. Enlargement of B.

Henning demonstrated on a widefield DeltaVision system that KASH5 localized to foci at the ends of the SC. Together with Graham, they set out to use 3D-SIM on the DeltaVision OMX to resolve the pair of axial elements within the SC. Henning recalls being amazed when he saw the first super resolution image he acquired with Graham because 3D-SIM clearly resolved the two AEs and revealed that KASH5 localizes as rings (not foci!) at the tip of each AE (Fig 1). The images they acquired went on to win an institute-wide image competition, the international GE Healthcare Image Competition in 2013, and impressively, were chosen for the cover of The Journal of Cell Biology.

With this new information, the authors shifted their focus to examine the localization of another protein in the same structural complex as KASH5, called SUN1. Again using 3D-SIM on the DeltaVision OMX, they found that SUN1, like KASH5, localizes in rings at the ends of the AE that form figure 8-like structures (Fig 1, white arrow) during homolog pairing.

In summary, these authors used 3D-SIM on the DeltaVision OMX to identify new structures associated with the SC that function to connect chromosomes to the cytoskeleton in meiosis.


3D-SIM played a key role in identifying novel structures of SUN1 and KASH5 in meiosis. In The Journal of Cell Biology publication, Henning and Graham note: “SIM is a valuable tool in our studies of spermatogenesis. The twofold improvement in resolution over conventional widefield microscopy allowed us to directly assess homolog pairing and synapsis by following the alignment of the SC axial elements.”

Featured Publications imaging the SC with DeltaVision OMX

Phillips, D. et al. High resolution analysis of meiotic chromosome structure and behavior in barley (Hordeum vulgare L.) PLoS One 7(6) (2012). doi:10.1371/journal. pone.0039539.

Zhang, W. et al. HAL-2 promotes homologous pairing during Caenorhabditis elegans meiosis by antagonizing inhibitory effects of synaptonemal complex precursors PLoS Genet. 8(8) (2012). doi:10.1371/journal. pgen.1002880.

Horn, H. et al. A mammalian KASH domain protein coupling meiotic chromosomes to the cytoskeleton J. Cell Biol. 202(7), 1023–39 (2013). doi:10.1083/jcb.201304004.

Sato-Carlton, A. et al. Protein phosphatase 4 promotes chromosome pairing and synapsis, and contributes to maintaining crossover competence with increasing age PLos Genet. 10(10) (2014). doi:10.1371/ journal.pgen.1004638.

Collins, K. et al. Corolla is a novel protein that contributes to the architecture of the synaptonemal complex of Drosophila Genetics 198(1), 219–28 (2014). doi:10.1534/ genetics.114.165290/-/DC1.

Lake, C. et al. Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila eLIFE e08287, 1-26 (2015). doi:10.7554/eLife.08287.

Lambing, C. et al. Arabidopsis PCH2 mediates meiotic chromosome remodeling and maturation of crossovers. PLOS Genet. 11(7), (2015), doi:10.1371/journal.pgen.1005372.

Subramanian, B. et al. Chromosome synapsis alleviates Mek1-dependent suppression of meiotic DNA repair. PLos Biol e1002369, 1-26 (2016), doi:10.1371/journal.pbio.1002369.

Colas, I. et al. A spontaneous mutation in MutL-Homolog 3 (HvMLH3) affects synapsis and crossover resolution in the barley desynaptic mutant des10. New Phytol (2016), doi:10.1111/nph.14061.

Rong, M. et al. Meiotic cohesion subunits RAD21L and REC8 are positioned at distinct regions between lateral elements and traverse filaments in the synaptonemal complex of mouse spermatocytes. J Reprod Dev (2016), Published online: September 26, 2016.


  1. Bolor, H. et al. Mutations of the SYCP3 Gene in Women with Recurrent Pregnancy Loss. AJHG 84(1), 14–20 (2009).
  2. Hassold, T., Hall, H., and Hunt, P. The origin of human aneuploidy: where we have been, where we are going. Hum. Mol. Genet. 16(2), 203–8 (2007).

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