In vivo two-photon imaging reveals dynamics and signaling in one-to-one pollen tube guidance

Abstract

Imaging and analysis of precise morphology and tissue structure are important for understanding the development of organisms and tissue structure. However, it is generally difficult to visualize the deep tissues of living organisms because the body of organisms is opaque. Two-photon excitation microscopy achieves deeper penetration and reduces photodamage compared to single-photon excitation, such as confocal microscopy. Therefore, it is widely used for in vivo live imaging. We have demonstrated the importance of selecting both appropriate fluorescent proteins and excitation wavelengths for clear two-photon imaging in deep plant tissues. We also developed a two-photon live imaging method, named single-locule method, to directly observe the process of pollen tube guidance and fertilization in the pistil of Arabidopsis thaliana. Spatiotemporal regulation of pollen tube growth by each cell of the female and male gametophyte and maternal tissues results in multistep polyspermy blockings that achieve successful fertilization. In addition to these recent studies, this review also provides tips on how to observe deep plant tissues in vivo and how the single-locule method was established.