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

## TL;DR

This paper introduces a new imaging technique that allows detailed, label-free observation of plant roots and microbes in their natural environment.

## Contribution

The study introduces THG microscopy as a novel, label-free method for imaging root structures and microbes with high resolution.

## Key findings

- THG imaging revealed root structures like vasculature, Casparian strips, and root cap cells.
- THG provided contrast for visualizing bacteria and fungi in the root rhizosphere without labels.
- Combined THG and 3PF imaging enabled tracking of microbes and subcellular features in the rhizosphere.

## 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 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.

The online version contains supplementary material available at 10.1038/s41598-025-20030-9.