High-resolution macro-to-micro 3D observation of cleared rice anthers : sample preparation in view of the refractive index of immersion media
Flowers have organs called anthers. This organ, also commonly called the “stamen”, has a bag-like structure in which pollen develops. Pollen formation is a process of a succession of generations accompanied by meiosis, and the generation and development of anthers as well as pollen have important biological implications. Anther generation and development have long been observed using optical and electron microscopes, and in recent years research has been conducted at the genetic and molecular level. Most recently, microRNA-mediated regulation of anther wall (anther sack) formation has been revealed and attracted attention (Komiya et al.).
One microscope observation technique that has been attracting attention in recent years is a technique for making transparent tissues, organs, and individuals. Techniques such as ClearSee, TOMEI, and PEA-CLARITY have been developed to make transparent plants, and ClearSeeAlpha (Kurihara et al.) and iTOMEI (Sakamoto et al.) are further developments thereof. These clearing reagents are often used in combination with confocal microscopy and multiphoton excitation microscopy, which are effective for the deep observation of samples.
Conventionally, anther development was observed in sections prepared using a microtome, but it is difficult to understand accurate three-dimensional structures from slices of two-dimensional images. In this respect, the combination of clearing method and confocal microscopy is considered to be very effective for 3D observation of deep sites. However, there have been problems concerning the degree of clearing and processing time of the clearing reagent differing according to the plant species and types of tissues/organs, as well as the inevitability of aberrations accompanying deep observation with a microscope.
This application note introduces an example of acquisition of 3D images of rice anthers under optimal optical conditions in order to understand their structures at the cellular level, by searching for the best clearing reagent, applying clearing, and mounting the samples with a solidified clearing reagent.