Imaging transzonal projections in the cumulus-oocyte complexes: challenges and solutions†.

Deep three-dimensional imaging of oocytes shows several difficulties. Their large size and spherical shape cause depth-dependent artefactual shadow in the middle, resulting from refractive index mismatches induced by turbid organelles and lipid droplets. These mismatches lead to optical aberrations, increasing the laser spot size at the confocal pinhole plan and causing significant attenuation of fluorescence intensity, making it difficult to clearly image fine structures such as the transzonal projections (TZPs) connecting cumulus cells and the oocyte. To overcome these challenges, various methods of sample preparation and confocal imagery settings were compared. To clearly show the depth limitation, a clearing protocol was used to image entire fixed embryos. As expected, limiting diffraction, namely, by removing lipid droplets and harmonizing extra- and intracellular media, resulted in more uniform staining and distribution, compared to uncleared specimens. The density of the cumulus cloud and fixation protocols were shown to have a profound impact on image quality. Gentle partial stripping and low fixation reduced noise in imagery, while permeabilization with Triton enhanced antibody penetration, resulting in efficient protein labeling with the zona pellucida-enclosed TZPs. Control samples were employed to exemplify unspecific and specific signals to determine optimal confocal settings. Careful consideration of confocal parameters was shown to be crucial for well-adjusted imagery. Moreover, the choice of mounting medium and slide assembly impacts the shape and resolution of the specimen. These findings provide valuable insights into challenges associated with cumulus-oocyte complex imaging, offering solutions for optimizing sample preparation and image quality.