Unbiased Complete Estimation of Chloroplast Number in Plant Cells Using Deep Learning Methods

Abstract

Chloroplasts are essential organelles in plants that are involved in plant development and photosynthesis. Accurate quantification of chloroplast numbers is important for understanding the status and type of plant cells, as well as assessing photosynthetic potential and efficiency. Traditional methods of counting chloroplasts using microscopy are time-consuming and face challenges such as the possibility of missing out-of-focus samples or double counting when adjusting the focal position. Here, we developed an innovative approach called Detecting- and-Counting-chloroplasts (D&Cchl) for automated detection and counting of chloroplasts. This approach utilizes a deep-learning-based object detection algorithm called You-Only-Look-Once (YOLO), along with the Intersection Over Union (IOU) strategy. The application of D&Cchl has shown excellent performance in accurately identifying and quantifying chloroplasts. This holds true when applied to both a single image and a three-dimensional (3D) structure composed of a series of images. Furthermore, by integrating Cellpose, a cell-segmentation tool, we were able to successfully perform single-cell 3D chloroplast counting. Compared to manual counting methods, this approach improved the accuracy of detection and counting to over 95%. Together, our work not only provides an efficient and reliable tool for accurately analyzing the status of chloroplasts, enhancing our understanding of plant photosynthetic cells and growth characteristics, but also makes a significant contribution to the convergence of botany and deep learning. One-sentence summary This deep learning-based approach enables the accurate complete detection and counting of chloroplasts in 3D single cells using microscopic image stacks, and showcases a successful example of utilizing deep learning methods to analyze subcellular spatial information in plant cells. The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors ( https://academic.oup.com/plcell/ ) is: Zhao Dong ( dongzhao@hebeu.edu.cn ), Shaokai Yang, ( shaokai1@ualberta.ca ), Ningjing Liu ( liuningjing1@yeah.net ), and Qiong Zhao ( qzhao@bio.ecnu.edu.cn ).

Code and data availability

The authors explicitly state that all code and the training dataset (annotated chloroplast microscopy images) are shared on their public GitHub repository, which is a paper-specific asset for this chloroplast counting study. Other URLs (labelImg, yolov7, ImageJ Falk plugins) are generic third-party tools, not paper-own