Label-free structural imaging of plant roots and microbes using third- harmonic generation microscopy

Abstract

ABSTRACT Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF fluorescence signal, we demonstrated our ability to investigate root-microbe interactions by achieving single-bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.

Plant phenotyping relevance

THG・3PFによる生根の構造と細胞内特徴を、ラベルフリーかつ生体内で可視化するイメージング手法が研究の中心であり、植物形態・状態の表現型取得に直接関与する。

Code and data availability

The paper's imaging data are stated to be contained only in the article and its supplementary files, with no public repository deposit, no author analysis code URL, and no trained model. The eco-fab.org link is a generic device-design resource, not a paper-specific dataset or code asset.

No evidence-backed public reproduction asset is currently recorded.