Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

During meiotic prophase, telomeres attach to the inner nuclear envelope and cluster to form the so-called meiotic bouquet. Although this has been observed in almost all organisms studied, its precise function remains elusive. The coincidence of telomere clustering and initiation of chromosome synapsis has led to the hypothesis that the bouquet facilitates homologous chromosome pairing and synapsis. However, recent mutant analysis suggests that the bouquet is not absolutely required for either homologous pairing or synapsis but that it makes both processes much faster and more efficient. The initiation of bouquet formation is independent of the initiation of recombination. However, the progression through recombination and synapsis may be required for exit from the bouquet stage. Little is known about the mechanism of telomere clustering but recent studies show that it is an active process.

REC8 is a master regulator of chromatin structure and function during meiosis. Here, we dissected the functions of absence of first division (afd1), a maize rec8/α-kleisin homolog, using a unique afd1 allelic series. The first observable defect in afd1 mutants is the inability to make a leptotene chromosome. AFD1 protein is required for elongation of axial elements but not for their initial recruitment, thus showing that AFD1 acts downstream of ASY1/HOP1. AFD1 is associated with the axial and later the lateral elements of the synaptonemal complex. Rescuing 50% of axial element elongation in the weakest afd1 allele restored bouquet formation demonstrating that extent of telomere clustering depends on axial element elongation. However, rescuing bouquet formation was not sufficient for either proper RAD51 distribution or homologous pairing. It provides the basis for a model in which AFD1/REC8 controls homologous pairing through its role in axial element elongation and the subsequent distribution of the recombination machinery independent of bouquet formation.

Pairing, synapsis, and recombination are prerequisites for accurate chromosome segregation in meiosis. The phs1 gene in maize is required for pairing to occur between homologous chromosomes. In the phs1 mutant, homologous chromosome synapsis is completely replaced by synapsis between nonhomologous partners. The phs1 gene is also required for installation of the meiotic recombination machinery on chromosomes, as the mutant almost completely lacks chromosomal foci of the recombination protein RAD51. Thus, in the phs1 mutant, synapsis is uncoupled from recombination and pairing. The protein encoded by the phs1 gene likely acts in a multistep process to coordinate pairing, recombination, and synapsis.