Kato Yuta, Oi Takao, Sato Yoshikatsu, Taniguchi Mitsutaka
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Institute of Transformative Bio-Molecules, Nagoya University, Nagoya, Japan.
Bio Protoc. 2025 Aug 5;15(15):e5404. doi: 10.21769/BioProtoc.5404.
In response to environmental changes, chloroplasts, the cellular organelles responsible for photosynthesis, undergo intracellular repositioning, a phenomenon known as chloroplast movement. Observing chloroplast movement within leaf tissues remains technically challenging in leaves consisting of multiple cell layers, where light scattering and absorption hinder deep tissue visualization. This limitation has been particularly problematic when analyzing chloroplast movement in the mesophyll cells of C plants, which possess two distinct types of concentrically arranged photosynthetic cells. In response to stress stimuli, mesophyll chloroplasts aggregate toward the inner bundle sheath cells. However, conventional methods have not been able to observe these chloroplast dynamics over time in living cells, making it difficult to assess the influence of adjacent bundle sheath cells on this movement. Here, we present a protocol for live leaf section imaging that enables long-term and detailed observation of chloroplast movement in internal leaf tissues without chemical fixation. In this method, a leaf blade section prepared either using a vibratome or by hand was placed in a groove made of a silicone rubber sheet attached to a glass slide for microscopic observation. This technique allows for the quantitative tracking of chloroplast movement relative to the surrounding cells. In addition, by adjusting the sectioning angle and thickness of the unfixed leaf sections, it is possible to selectively inactivate specific cell types based on their size and shape differences. This protocol enables the investigation of the intercellular interactions involved in chloroplast dynamics in leaf tissues. Key features • Thin leaf sections prepared while still alive enable prolonged microscopic observation of chloroplast movement within the leaf tissue. • Selective cell inactivation can be achieved by adjusting the slice thickness and angle. • This method is applicable to a wide range of plant species.
为响应环境变化，叶绿体（负责光合作用的细胞器）会在细胞内重新定位，这一现象被称为叶绿体运动。在由多层细胞组成的叶片中，观察叶片组织内的叶绿体运动在技术上仍具有挑战性，因为光散射和吸收会阻碍对深层组织的可视化。当分析C植物叶肉细胞中的叶绿体运动时，这一限制尤其成问题，因为C植物拥有两种不同类型的同心排列的光合细胞。在应激刺激下，叶肉叶绿体向内部的维管束鞘细胞聚集。然而，传统方法无法在活细胞中长时间观察这些叶绿体动态，因此难以评估相邻维管束鞘细胞对这种运动的影响。在此，我们提出一种活叶切片成像方案，该方案能够在不进行化学固定的情况下，对叶片内部组织中的叶绿体运动进行长期且详细的观察。在这种方法中，使用振动切片机或手工制备的叶片切片被放置在附着于载玻片的硅橡胶片制成的凹槽中，用于显微镜观察。该技术允许对叶绿体相对于周围细胞的运动进行定量跟踪。此外，通过调整未固定叶片切片的切片角度和厚度，可以根据特定细胞类型的大小和形状差异选择性地使其失活。该方案能够研究叶片组织中叶绿体动态所涉及的细胞间相互作用。关键特性 • 仍保持存活状态时制备的薄叶片切片能够对叶片组织内的叶绿体运动进行长时间的显微镜观察。 • 通过调整切片厚度和角度可以实现选择性细胞失活。 • 该方法适用于多种植物物种。
