This protocol describes the fluorescence in situ hybridization (FISH) of DNA probes on mitotic chromosome spreads optimized for two super-resolution microscopy approaches-structured illumination microscopy (SIM) and stimulated emission depletion (STED). It is based on traditional DNA FISH methods that can be combined with immunofluorescence labeling (Immuno-FISH). This technique previously allowed us to visualize ribosomal DNA linkages between human acrocentric chromosomes and provided information about the activity status of linked rDNA loci. Compared to the conventional wide-field and confocal microscopy, the quality of SIM and STED data depends a lot more on the optimal specimen preparation, choice of fluorophores, and quality of the fluorescent labeling. This protocol highlights details that make specimens suitable for super-resolution microscopy and tips for good imaging practices.

OBJECTIVE: The purpose of this report is to analyze the chromosome status and fertilization capability of sperm obtained from an infertile male patient with ring chromosome 15.
METHODS: This was a case report at a private in vitro fertilization clinic. A man diagnosed with severe oligozoospermia carrying ring chromosome 15. To evaluate the chromosome status and fertilization capability, sperm from a patient carrying ring chromosome 15 were injected into enucleated mouse oocytes.
RESULTS: The karyotypes of motile sperm from a patient carrying ring chromosome 15 were normal, and ring chromosome 15 was not observed in the chromosome spread samples of 1PN. In addition, these motile sperm retained the fertilization capability. However, the fertilization rates decreased (85.2, 76.2, and 64.3%, respectively) along with the decline of the aspect ratio of the sperm head (≥ 1.50, 1.30-1.49, and < 1.30, respectively).
CONCLUSIONS: The karyotypes were normal without ring chromosome 15, and motile sperm with a high aspect ratio showed adequate potential for fertilization.

Significant advances in chromosome preparation and other techniques have greatly increased the potential of plant cytogenetics in recent years. Increase in longitudinal resolution using DNA extended fibers as well as new developments in imaging and signal amplification technologies have enhanced the ability of FISH to detect small gene targets. The combination of fluorescence in situ hybridization with immunocytochemistry allows the investigation of cell events, chromosomal rearrangements and chromatin features typical for plant nuclei. Chromosome manipulation techniques using microdissection and flow sorting have accelerated the analysis of complex plant genomes. Together, the different cytogenetic approaches are invaluable for the unravelling of detailed structures of plant chromosomes, which are of utmost importance for the study of genome properties, DNA replication and gene regulation. In this technical review, different cytogenetic approaches are discussed for the analysis of plant chromosomes, with a focus on mitotic chromosomes.