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

Area of Science:

• Plant Biology
• Microbiology
• Microscopy

Background:

• Root biology is crucial for agriculture and climate change mitigation but remains understudied due to imaging challenges.
• Existing methods struggle to visualize root structures and microbial interactions in their natural environment.

Purpose of the Study:

• To develop and apply a novel, label-free imaging technique for in situ visualization of plant roots and rhizosphere microbes.
• To enable high spatiotemporal resolution imaging of root structures and root-microbe interactions.

Main Methods:

• Utilized microfabricated ecosystems (EcoFABs) to create controlled growing environments with optical access.
• Employed nonlinear multimodal microscopy, specifically third-harmonic generation (THG) and three-photon fluorescence (3PF).
• Achieved label-free, in situ imaging of live roots and microbes at high resolution.

Main Results:

• THG microscopy visualized key root structures (vasculature, Casparian strips, meristems, root cap) and subcellular features (nuclear envelopes, nucleoli, starch granules).
• Label-free THG contrast was achieved for bacterial and fungal cell walls in the rhizosphere.
• Simultaneous 3PF imaging enabled single-bacterium tracking and subcellular visualization of fungal spores and hyphae.

Conclusions:

• The developed THG and 3PF microscopy technique provides unprecedented label-free, in situ imaging capabilities for root biology.
• This approach facilitates detailed investigation of root structure and complex root-microbe interactions in the rhizosphere.
• EcoFABs combined with advanced microscopy offer a powerful platform for advancing plant and microbial research.