An Optimized Optical Clearing Pipeline for Intact 3D Plant Imaging and Visualization of Fluorescent Probes
An Optimized Optical Clearing Pipeline for Intact 3D Plant Imaging and Visualization of Fluorescent Probes
复制标题
用于完整 3D 植物成像和荧光探针可视化的优化光学透明管道
DOI：
10.1093/micmic/ozad067.499
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发表时间：
2023
影响因子：
2.8
通讯作者：
Sanders, Mark A
中科院分区：
文献类型：
--
作者：
Solhaug, Erik;Roy, Rahul;Willey, Patrick T;Kane, Nadia;Carter, Clay;Sanders, Mark A
在这里，我们报告了针对植物组织优化的ClearSee1组织清除技术的改进版本，该技术使整个植物成像能够可视化染色和表达的荧光蛋白。这种方法促进了蛋白质和维管系统在完整组织中的3D定位，同时保留了细胞水平的结构，允许探索植物连接体的相互作用。植物组织的固定和嵌入用于分子询问，历史上依赖于组织学染色和免疫组织化学等技术作为植物细胞生物学研究的基础，这需要对组织切片、标记切片、成像每个切片，然后将图像重组为感兴趣结构的3D表示。在固定和组织清除技术方面的最新进展，如PEGASOS、SeeDB1、ScaleA22、IDISCO和X-Clarity，使动物器官、胚胎、大脑和其他器官的完整成像成为可能。然而，对完整的植物组织和器官的3D成像却没有引起太多的关注。在这里，我们提出了从切片重建到组织透明和自发荧光减少的根本转变，允许完整的植物3D可视化，同时保留感兴趣的内源分子结构。植物通过韧皮部(筛分元素)从来源组织(如叶片)运输糖和其他有机化合物，而水和无机离子通过木质部2移动。维管系统如何连接下沉组织，如根、花和种子，与装卸溶质的机制密切相关。在这项研究中，我们利用了表达韧皮部定位荧光蛋白的完整的拟南芥幼苗和组织，包括叶、冠(茎和花的组合)、花(单个开放的花)、角果(不同发育阶段的绿色豆荚)和根。我们的目标是优化清除程序，同时保留在转基因植物中表达的内源GFP和RFP蛋白的荧光。此外，我们的目标是鉴定这些表达的韧皮部蛋白与木质部的相互作用，以便在体内和原位进行比较成像。为此，在掺杂了罗丹明WT的土壤中种植了表达荧光蛋白的植物。在适当的阶段，植物被收获，固定，清理，安装在折射率匹配的介质中，并使用带有适当浸泡光学的激光扫描共聚焦显微镜成像，以验证各种处理步骤。我们将详细介绍所使用的技术，包括最佳化学固定、组织清除剂和时间、手头成像系统的最佳折射率匹配、成像条件以及我们共享研究设施中使用的数据管理管道。这使得我们能够在整个完整的组织中与罗丹明WT标记同时可视化表达的荧光蛋白，包括GFP和RFP，同时将结构和分子完整性的损失降至最低。随着研究不断加强对完整生物组织的清除、标记、成像和可视化方法，成像、分析和计算技术也将如此。在同心协力下，植物生物系统的分子和光学图谱在不久的将来将会有更多的应用。
Here we report an adapted version of the ClearSee1 tissue clearing technique optimized for plant tissues, which enables whole plant imaging to visualize stains and expressed fluorescent proteins. This approach facilitates protein and vascular system localization in intact tissue in 3D, while retaining cellular-level structure, permitting the exploration of plant connectome interactions. Fixation and embedding of plant tissue for molecular interrogation has historically relied on techniques such as histological staining and immunohistochemistry as the foundation of plant cell biology studies, which required sectioning the tissue, labeling the section, imaging each section and then reassemble the images into a 3D representation of the structures of interest. Recent advances in fixation and tissue clearing techniques such as PEGASOS, SeeDB1, ScaleA22, iDISCO, and X-CLARITY have enabled intact imaging of animal organoids, embryos, brains and other organs. However, less attention has been given to the 3D imaging of intact plant tissues and organs. Here we present a fundamental shift from section reconstruction to tissue clearing and autofluorescence reduction, allowing for intact plant 3D visualization while retaining endogenous molecular structures of interest.Plants transport sugars and other organic compounds from source tissues, like leaves, via the phloem (sieve elements), whereas water and inorganic ions move through the xylem2. How the vasculature connects to sink tissues, like roots, flowers and seeds, is intimately linked to the mechanism through which solutes are loaded and unloaded2. In this study we utilized intact young plants and tissues from Arabidopsis thaliana expressing phloem-localized fluorescent proteins, including leaves, crowns (combination of stems and flowers), flowers (individual open flowers), siliques (green seed pods at varied developmental stages) and roots. Our goal was to optimize the clearing protocol while retaining fluorescence of endogenous GFP and RFP proteins expressed in genetically modified plants. Additionally, we aimed to identify the interactions of these expressed phloem proteins with the xylem for comparative imaging in vivo and in situ. For this purpose, plants expressing fluorescent proteins were grown in soils doped with Rhodamine WT. At the appropriate stages, the plants were harvested, fixed, cleared, mounted in refractive index matching media, and imaged using a laser scanning confocal microscope with appropriate immersion optics to validate the various treatment steps. We will present the details of the techniques used, describing the optimal chemical fixation, tissue clearing reagents and times, optimal refractive index matching for the imaging systems at hand, imaging conditions, and data management pipelines used in our shared research facility. This has allowed us to visualize expressed fluorescent proteins, including GFP and RFP, simultaneously with Rhodamine WT labeling in whole intact tissues, while minimizing loss of structural and molecular integrity. As studies continue to strengthen clearing, labeling, imaging and visualization methods for intact biological tissues, so too will imaging, analysis, and computational technologies. With concerted and collaborative efforts, molecular and optical profiling of plant biological systems will find many more applications in the near future.