Harline Kate, Roeder Adrienne H K
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, 14853, USA.
Section of Plant Biology, School of Integrative Plant Sciences, Cornell University, Ithaca, NY, 14853, USA.
Plant Methods. 2023 Feb 1;19(1):10. doi: 10.1186/s13007-023-00987-2.
Live imaging is the gold standard for determining how cells give rise to organs. However, tracking many cells across whole organs over large developmental time windows is extremely challenging. In this work, we provide a comparably simple method for confocal live imaging entire Arabidopsis thaliana first leaves across early development. Our imaging method works for both wild-type leaves and the complex curved leaves of the jaw-1D mutant.
We find that dissecting the cotyledons, affixing a coverslip above the samples and mounting samples with perfluorodecalin yields optimal imaging series for robust cellular and organ level analysis. We provide details of our complementary image processing steps in MorphoGraphX software for segmenting, tracking lineages, and measuring a suite of cellular properties. We also provide MorphoGraphX image processing scripts we developed to automate analysis of segmented images and data presentation.
Our imaging techniques and processing steps combine into a robust imaging pipeline. With this pipeline we are able to examine important nuances in the cellular growth and differentiation of jaw-D versus WT leaves that have not been demonstrated before. Our pipeline is approachable and easy to use for leaf development live imaging.
实时成像技术是确定细胞如何形成器官的金标准。然而，在较大的发育时间窗口内对整个器官中的多个细胞进行追踪极具挑战性。在本研究中，我们提供了一种相对简单的方法，用于对拟南芥早期发育阶段的第一片叶子进行共聚焦实时成像。我们的成像方法适用于野生型叶片以及jaw-1D突变体的复杂弯曲叶片。
我们发现，解剖子叶、在样品上方固定盖玻片并使用全氟萘烷固定样品，可获得用于强大的细胞和器官水平分析的最佳成像系列。我们在MorphoGraphX软件中提供了互补图像处理步骤的详细信息，用于分割、追踪谱系以及测量一系列细胞特性。我们还提供了我们开发的MorphoGraphX图像处理脚本，用于自动分析分割图像和数据呈现。
我们的成像技术和处理步骤结合成了一个强大的成像流程。通过这个流程，我们能够研究jaw-D叶片与野生型叶片在细胞生长和分化方面以前未被证实的重要细微差别。我们的流程对于叶片发育实时成像来说易于使用且容易上手。
