Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip
Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip
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DOI：
10.3791/4290
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发表时间：
2012-07-01
影响因子：
1.2
通讯作者：
Frommer, Wolf B.
中科院分区：
文献类型：
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作者：
Grossmann, Guido;Meier, Matthias;Frommer, Wolf B.
根的功能是植物的物理锚，是负责吸收水分和矿物质营养物质的器官，如氮、磷、硫酸盐和植物从土壤中获得的微量元素。如果我们想开发可持续的方法来生产高产量的作物，我们需要更好地了解根系是如何发育的，如何吸收广泛的营养物质，以及如何与共生和致病生物相互作用。为了实现这些目标，我们需要能够在几分钟到几天的时间内，从微观的细节上探索树根。我们开发了RootChip，一种基于聚二甲基硅氧烷（PDMS）的微流体装置，它允许我们从拟南芥幼苗中生长和成像根，同时避免在成像准备过程中对根产生任何物理压力(1)（图1）。该装置包含一个分叉通道结构，该结构具有微机械阀，用于引导流体从溶液入口流向八个观察室(2)。这种灌注系统可以精确和快速地控制和修改根微环境。腔室的体积约为400升，因此只需要最少量的测试溶液。在这里，我们提供了一个详细的协议，研究根系生物学在RootChip上使用基于成像的方法与实时分辨率。用延时显微镜可以在几天内分析根部。根可以灌注营养液或抑制剂，多达八个幼苗可以并行分析。该系统具有广泛应用的潜力，包括在化学物质存在或不存在的情况下分析根生长，基于荧光的基因表达分析，以及生物传感器分析，例如FRET纳米传感器(3)。
The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.We developed the RootChip, a polydimethylsiloxane (PDMS)-based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging(1) (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers(2). This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors(3).