Live Leaf-Section Imaging for Visualizing Intracellular Chloroplast Movement and Analyzing Cell-Cell Interactions.

Abstract

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 4 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.

Plant phenotyping relevance

生葉切片のライブイメージングにより、葉内部の葉緑体運動を長時間観察・定量追跡する手法を開発しており、植物表現型の取得が研究の中心である。

Code and data availability

This Bio-protocol article describes live leaf-section imaging for chloroplast movement but provides no public phenotype datasets, author image/annotation data, analysis code, or trained models. The only URLs are the CC BY-NC license, a commercial red filter product page, the generic Fiji/ImageJ software site, and the (

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