To establish a 4D time-lapse system of Arabidopsis embryogenesis, we have optimized image acquisition for observation of live embryos under the microscope, we tested various microscopes, (1-photon confocal, 2-photon microscopes, light sheet), imaging settings, medium for ovule culture, tool to fix ovules during culture and marker lines that give an appropriate plasma membrane signal enabling 3D segmentation of obtained images. To be able to 3D-segment the images acquired in the time-lapse experiments, we tested various procedures for 3D segmentation in MorphographX, as well as the machine learning-based image processing software ilastik. To further improve the quality of obtained images, we tested deconvolution software. To develop a protocol to observe cellulose, we tested the use of a super resolution microscope, and worked with computational biologists to create custom solutions for quantification of cellulose fibers. To observe PHB:GFP expression, several marker lines were observed both in Arabidopsis and in Cardamine, and the lines that showed strong expression were selected. To perform laser ablation in embryo, the settings for ablation were tested. To perform 4D time-lapse in Cardamine, we screened for optimal plasma membrane marker lines and settings for imaging.
To develop computational models of embryo development we collaborated with Dr. Smith’s group, resulting in a detailed 2D growing model of early embryogenesis as well as 3D pressurized templates of early embryonic developmental stages. These models are used to better understand the interaction between growth, gene expression and cell mechanics.
Taken together, the protocols we developed will provide tools to better understand how combination of orientated cell division, cell growth and cell fate determination drive embryonic patterning and establish axes in different Brassicaceae spices.
The results of this project were presented/discussed by the researcher (1 conference, 2 workshops) and collaborators (1 conference). The researcher published two publications related to the subject/techniques required in this project. Publication from this project itself is currently under preparation.
