Lightsheet microscopy for studies of plant-environment interactions

Session

Stream 6 (Frontiers): Lightsheet Microscopy: Development and Application

Authors

Mr yangminghao liu (4), Dr Daniel Patko (3, 1), Dr Ilonka Engelhardt (3, 1), Dr Qizhi Yang (5), Dr Timothy S George (3), Dr Nicola Stanley-Wall (4), Dr Vincent Ladmiral (5), Dr Bruno Ameduri (5), Dr Tim J Daniell (3), Dr Nicola Holden (2), Dr Michael P MacDonald (4), Dr Lionel X Dupuy (1)

Affiliations

1. Department of Conservation of Natural Resources
2. Scotland's Rural College
3. The James Hutton Institute
4. University of Dundee
5. University of Montpellier

Keywords

lightsheet microscopy, plant-microbe interaction, environmental imaging.

Abstract text

Lightsheet microscopy enabled imaging of biological activity. We show a lightsheet microscope system capable of tracking bacterial movements during colonisation of the rhizosphere and of revealing soil chemical changes induced during plant growth. The development of a new generation of environmental microscope could greatly enhance our understanding of biological processes at ecological scale.

Plant growth is supported by many biophysical interactions with microorganisms, geochemicals, water and gas, within the complex soil-atmosphere physical environment. Unfortunately, observations of plants' interactions with both the biotic and abiotic environments are difficult to make at the microscopic scale^1–3, as they occur throughout large volumes of influence and natural soils are opaque, creating a severe challenge for conventional microscopy.

A custom-made, multispectral lightsheet microscope was developed to resolve live plant-environment interactions across entire seedlings, in mesocosms comprising a glass chamber of a few cm³ volume and transparent soil. This system was used to track microbial movements and interactions with the soil pore and plant roots using lettuce plants and GFP tagged Bacillus Subtilis. We also demonstrate application of the microscope to measure pH changes during the growth of lettuce plants using newly developed pH sensitive soil. Various image processing and image analysis tools were also developed to quantify including flat field correction, image stitching, and Neural Network models for pH prediction in soil.

The microscope revealed previously unseen patterns of microbial activity in soil. Bacillus Subtilis were shown to favour small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. the soil pore structure influenced the behaviour of the bacteria, both before and during the formation of biofilms on the root surface something which could not have been observed with experiments run in liquid cultures^4-6. Migrations appeared to be directed first towards the root cap before subsequent colonisation of mature epidermis cells. The microscope could also reveal plant-induced acidification of soil particles at the microscopic scale, which far exceed the ability of optochemical sensors currently in use.

When combined with environment sensing transparent soil, lightsheet microscopy enables fast in situ observation of biological process. The study demonstrates an ability to observe both the plant and micro-organisms within their structurally complex environments. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand life in soils

References

1.        Sasse, J., Martinoia, E. & Northen, T. Feed Your Friends: Do Plant Exudates Shape the Root Microbiome? Trends Plant Sci. 23, 25–41 (2018).

2.        Dupuy, L. X. & Silk, W. K. Mechanisms of Early Microbial Establishment on Growing Root Surfaces. Vadose Zo. J. 15, vzj2015.06.0094 (2016).

3.        Deng, J. et al. Synergistic effects of soil microstructure and bacterial EPS on drying rate in emulated soil micromodels. Soil Biol. Biochem. 83, 116–124 (2015).

4.        Massalha, H., Korenblum, E., Malitsky, S., Shapiro, O. H. & Aharoni, A. Live imaging of root–bacteria interactions in a microfluidics setup. Proc. Natl. Acad. Sci. 114, 4549–4554 (2017).

5.        Noirot-Gros, M.-F. et al. Functional Imaging of Microbial Interactions With Tree Roots Using a Microfluidics Setup. Front. Plant Sci. 11, (2020).

6.        Aufrecht, J. A. et al. Quantifying the Spatiotemporal Dynamics of Plant Root Colonization by Beneficial Bacteria in a Microfluidic Habitat. Adv. Biosyst. 2, 1800048 (2018).