Accelerating Automated Stomata Analysis Through Simplified Sample Collection and Imaging Techniques

Luke Millstead; Hiranya Jayakody; Harsh Patel; Vihaan Kaura; Paul R Petrie; Florence Tomasetig; Mark Whitty

doi:10.3389/fpls.2020.580389

Accelerating Automated Stomata Analysis Through Simplified Sample Collection and Imaging Techniques

Front Plant Sci. 2020 Sep 25:11:580389. doi: 10.3389/fpls.2020.580389. eCollection 2020.

Authors

Luke Millstead¹, Hiranya Jayakody¹, Harsh Patel¹, Vihaan Kaura¹, Paul R Petrie^{1

2}, Florence Tomasetig³, Mark Whitty¹

Affiliations

¹ School of Mechanical and Manufacturing Engineering, University of New South Wales, Sydney, NSW, Australia.
² Crop Sciences Division, South Australian Research and Development Institute, Waite Campus, Urrbrae, SA, Australia.
³ Mark Wainwright Analytical Centre, University of New South Wales, Sydney, NSW, Australia.

Abstract

Digital image processing is commonly used in plant health and growth analysis, aiming to improve research efficiency and repeatability. One focus is analysing the morphology of stomata, with the aim to better understand the regulation of gas exchange, its link to photosynthesis and water use and how they are influenced by climatic conditions. Despite the key role played by these cells, their microscopic analysis is largely manual, requiring intricate sample collection, laborious microscope application and the manual operation of a graphical user interface to identify and measure stomata. This research proposes a simple, end-to-end solution which enables automatic analysis of stomata by introducing key changes to imaging techniques, stomata detection as well as stomatal pore area calculation. An optimal procedure was developed for sample collection and imaging by investigating the suitability of using an automatic microscope slide scanner to image nail polish imprints. The use of the slide scanner allows the rapid collection of high-quality images from entire samples with minimal manual effort. A convolutional neural network was used to automatically detect stomata in the input image, achieving average precision, recall and F-score values of 0.79, 0.85, and 0.82 across four plant species. A novel binary segmentation and stomatal cross section analysis method is developed to estimate the pore boundary and calculate the associated area. The pore estimation algorithm correctly identifies stomata pores 73.72% of the time. Ultimately, this research presents a fast and simplified method of stomatal assay generation requiring minimal human intervention, enhancing the speed of acquiring plant health information.

Keywords: high-throughput analysis; microscope imagery; stomata analysis pipeline; stomata pore measurement; stomata sample collection.