Cells without borders: Optimization of Oligo-FiSH for maize meiocytes

Fluorescent in situ hybridization (FiSH) using whole-chromosome oligonucleotide
paints is a powerful cytogenetic technique for visualizing chromosomes and their behavior during cell division. This method enables the detection of chromosomal abnormalities such as non-homologous pairing and aberrant recombination events during meiosis. While traditional FiSH uses labeled DNA or RNA probes targeting specific chromosomal regions, Oligo-FiSH employs complex libraries of fluorescently labeled oligonucleotides to “paint” entire chromosomes with high specificity and resolution. Despite its potential, Oligo-FiSH requires careful optimization in plants, where rigid cell walls and dense cytoplasm cause significant challenges. The cell wall limits probe penetration, and the cytoplasm often produces autofluorescence, both of which reduce signal quality. Enzymatic digestion is commonly used to overcome these barriers; however, overdigestion can lead to fragile or degraded nuclei that are easily lost during slide preparation, while underdigestion leaves cytoplasmic material that interferes with probe hybridization. In this study, we optimized slide preparation protocol for Oligo-FiSH on maize meiocytes from both maize diploid and tetraploid lines. Meiocytes were dissected from fixed anthers, and various preparation strategies were tested to minimize cell loss and improve the quality of chromosome spreads. Our goal was to obtain cytoplasm-free, structurally intact chromosomes at late pachytene suitable for high-quality Oligo-FiSH imaging. Using this optimized method, we successfully visualized specific chromosome pairs in both diploid and polyploid maize meiocytes. Further analysis of meiotic abnormalities in polyploid lines will be the focus of our next research phase.

The internship program at the Boyce Thompson Institute gave me an amazing opportunity to grow professionally and personally. I really enjoyed working with other students in the program and learning from the professors at the institute. One of the highlights of my experience was working in Prof. Wojtek Pawlowski’s lab. Under the mentorship of Olga Zimina, I developed key skills in plant cytogenetics, including FiSH and advanced microscopy techniques. Beyond technical training, she encouraged me to become more independent in the lab guiding me to consult the literature and take initiative in refining protocols. With her support, I was able to optimize the Oligo-FiSH protocol, which significantly improved slide quality and allowed for the collection of more high-quality images. I am deeply grateful to my lab for their guidance and support. This experience has strengthened my passion for science and confirmed my desire to pursue a career in research and academia.