Three-dimensional reconstruction of Picea wilsonii Mast. pollen grains using automated electron microscopy
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Three-dimensional electron microscopy (3D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3D reconstruction of biological samples remains challenging. Herein, we present a 3D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and 38.57 nm lateral resolution using AutoCUTS-SEM (automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P. wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.
Keywords3D reconstruction AutoCUTS-SEM electron microscopy pollen
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We gratefully acknowledge Xi Chen from the Institute of Automation, Chinese Academy of Sciences for the help provided with image alignment and distortion correction, and Libo Jiang from the College of Biological Sciences & Biotechnology, Beijing Forestry University for discussions. This work was supported by grants from Fundamental Research Funds for the Central Universities (BLX201617), the Program of Introducing Talents of Discipline to Universities (111 projects, B13007), and the National Natural Science Foundation of China (31700250, 31530084, 31761133009, 31670182). This research was also supported by the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences.
Compliance and ethics The author(s) declare that they have no conflict of interest.
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