拟南芥根部核形和染色质动态的定量活细胞成像
2026年6月5日
该过程结合共軛焦显微镜配合拟 南芥 中荧光标记的核和染色质标记线，同时记录拟 南芥 根部的核和染色质动态，并研究这些动态随发育和环境信号变化。
我们的研究探讨了能够长期从渗透胁迫中恢复根系的基因和机制，以增强作物抵御干旱的能力。虽然该方案是为拟南芥根部开发的，但该方案可适应具有荧光蛋白标记系的系统。获取含有拟南芥幼苗的生长板，准备成像。
将60微升纯水移液器移入玻璃载玻片上。将三根六天大A. thaliana幼苗根放入水中。在根部上放置一个22x22毫米的1.5毫米号盖膜，但不覆盖叶片。
在共聚焦显微镜设置中，将激光曝光时间设置为1000毫秒。在激光合路器设置中，将488纳米激光设置为50%，用于绿色荧光蛋白（GFP）成像。将561纳米激光器设置为50%，用于红光蛋白（RFP）成像。
使用20倍物镜（不含光学变焦）成像样品。将Z切片步长设为0.3微米。将成像间隔设为一分钟，总成像时长为10分钟。
找到根，选择最多20个Z切片。捕捉16位图像。开始时间流逝采集，并用无损压缩保存为TIFF文件。
将收集到的数据转移到计算机进行分析。打开ImageJ或Fiji软件。点击并拖动 VSI 文件到 ImageJ 或 Fiji 应用主窗口。
在“生物格式导入选项”提示中，点击右下角的确定。然后在简介格式系列选项提示中，确保选择了系列1。点击左下角的确定。
图像加载完成后，通过选择图像来分割信号。选择颜色并选择分流通道。查看为GFP标记的核和RFP标记的染色质生成的独立窗口。
确定两个图像窗口中哪个对应染色质。点击染色质图像窗口后，通过选择图像，选择堆栈，然后从主 ImageJ 窗口点击 Z 项目来执行 Z 项目操作。把投影类型改成最大强度。
确保所有时间框架处于激活状态，并点击确定生成预测。通过按 Control Shift C 打开对比菜单，或选择图片并选择调整，然后从主 ImageJ 窗口点击亮度或对比度。将最小值横杆向左滑动，直到染色质清晰可见。
进入插件。点击插件菜单中的向下箭头，滚动到追踪选项。选择“追踪”并点击“TrackMate”。
确认校准设置准确，然后点击“下一步”。选择阈值检测器并点击“下一步”。切换到图像窗口延时的最后一帧。
点击强度阈值旁边的自动，然后点击预览。调整强度阈值，点击预览。重复调整和预览过程，直到所有染色质都被包含且噪音最小，然后点击“下一步”。
等待初始阈值检测完成，然后点击“下一步”。手动调整阈值直到只可见染色质，然后点击“下一步”。调整荧光图像显示并点击“下一步”。
选择“简单LAP追踪器”并点击“下一步”。将连接最大距离设置为5微米，最大闭合距离为15微米，最大闭合距离为1，然后点击下一步。让追踪完成。
观察原始图像窗口中显示的轨道，然后点击“下一步”。点击左下角的绿色加号按钮来创建筛选。在赛道模式中选择数量。
调整阈值滑块。确保过滤模式设置为“以上”，然后点击“下一步”。点击显示选项页面底部的“轨道”按钮。
选择导出为CSV。忽略内置的绘图页面，点击“下一步”。查看TrackMate分析结果。
点击“捕获叠加层”，然后选择执行。通过选择“文件”并点击“另存为”，然后选择GIF来保存视频。盐处理的根细胞表现出应激，着丝粒焦点比对照条件更大。
对照条件下染色质速度显著高于100毫莫拉氯化钠处理条件下。该方案能够追踪和定量拟南芥根中荧光标记蛋白随时间的变化。该方案中最重要的挑战是获得高质量图像，因为光漂白和样品漂移会使追踪变得困难。
未来的研究应探究我们研究中观察到的染色质运动减少的机制。
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Overview
The nucleus in eukaryotic cells exhibits dynamic shape changes, which are altered under stress conditions such as cancer in animals or abiotic stress in plants. This study developed a live-cell imaging pipeline in Arabidopsis thaliana roots using dual fluorescent markers for the nuclear envelope and chromatin to quantify nuclear and chromatin dynamics under control and salt-stressed conditions. Confocal microscopy combined with Fiji/ImageJ and TrackMate enabled quantitative analysis, revealing reduced chromatin movement under salt stress. The method provides a robust approach to study how abiotic stressors affect nuclear architecture and chromatin dynamics in plant cells.
Key Study Components
Area of Science
- Plant cell biology
- Nuclear dynamics
- Chromatin mobility
- Abiotic stress response
Background
- Nuclear shape is dynamic in live cells, not static as traditionally depicted.
- Altered nuclear morphology is associated with cancer in animal cells and environmental stress in plants.
- The nuclear envelope interacts with chromatin to influence gene expression.
- Limited knowledge exists on how abiotic stress affects nuclear shape, movement, and chromatin dynamics in plants.
Purpose of Study
- To develop a pipeline for quantifying nuclear and chromatin dynamics in live plant cells.
- To apply this method to assess changes under abiotic stress, specifically salt stress.
- To validate the approach by measuring chromatin movement in control and stressed conditions.
- To enable future analysis of nuclear and chromatin responses to environmental stressors in Arabidopsis roots.
Methods Used
- Live-cell imaging of Arabidopsis thaliana roots using confocal microscopy.
- Use of a dual fluorescently tagged marker line (nuclear envelope protein and chromatin).
- Image acquisition under control and salt-stressed conditions.
- Analysis of nuclear and chromatin dynamics using Fiji/ImageJ with the TrackMate plugin.
- Particle tracking to quantify chromatin speed and movement.
Main Results
- Chromatin movement was significantly reduced in salt-stressed roots compared to control.
- The dual-tagging system allowed simultaneous visualization of nuclear envelope and chromatin.
- TrackMate enabled accurate particle tracking and quantification of dynamics.
- The method successfully captured live, quantitative changes in nuclear and chromatin behavior.
Conclusions
- The developed pipeline allows for quantitative live-cell imaging of nuclear shape and chromatin dynamics in plant roots.
- Salt stress leads to decreased chromatin mobility, indicating altered nuclear dynamics under abiotic stress.
- This approach can be applied to study nuclear responses to various environmental stressors.
- The method provides a foundation for linking nuclear architecture to gene regulation and stress adaptation in plants.
Frequently Asked Questions
Executive Industry Relevance
Quantitative live-cell imaging of nuclear shape and chromatin dynamics in Arabidopsis roots provides a robust platform for dissecting cellular responses to environmental stress at high resolution. This approach enables predictive confidence in linking nuclear architecture changes to gene regulation, supporting early discovery and mechanistic de-risking in plant biotechnology pipelines. The method's quantitative outputs facilitate risk-adjusted decisions for trait development and stress adaptation research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of nuclear and chromatin behavior under defined stress conditions.
- Supports mechanistic de-risking by quantifying dynamic nuclear responses to abiotic stimuli.
- Facilitates functional validation of nuclear envelope and chromatin-associated targets.
Screening & Assay Development
- Provides standardized, quantitative imaging assays for nuclear and chromatin dynamics.
- Enables reproducible measurement of stress-induced changes in live plant cells.
- Supports scalable screening of genetic or chemical modulators affecting nuclear architecture.
Translational & Preclinical Research
- Aligns nuclear and chromatin dynamics with stress adaptation phenotypes in model systems.
- Enables continuity from discovery to preclinical validation of stress resilience traits.
- Supports predictive de-risking for translational research in crop improvement.
Pipeline & Workflow Integration
This imaging and analysis pipeline integrates from early discovery through screening and translational research in plant biotechnology.
- Discovery Biology: Quantifies nuclear and chromatin dynamics to clarify stress response pathways.
- Screening: Delivers reproducible, quantitative readouts for assay development and compound evaluation.
- Analytics: Provides particle tracking and statistical outputs for robust condition comparison.
- Translational Research: Links nuclear architecture changes to stress adaptation, supporting trait validation.
- Enterprise Reuse: Offers a reusable imaging and analysis workflow adaptable to diverse plant models and stressors.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in nuclear and chromatin target validation.
- Operational Value: Standardizes live-cell imaging and quantitative analysis for reproducibility.
- Strategic Value: Improves go/no-go decisions for stress adaptation trait development.
- Portfolio Impact: Enables risk-adjusted prioritization of genetic or chemical interventions.
Implementation Considerations
- Requires expertise in confocal microscopy and live-cell imaging.
- Needs access to dual fluorescent marker lines and image analysis software (Fiji/ImageJ, TrackMate).
- Demands cross-team standardization for imaging protocols and data analysis.
- Adaptable to other plant species with suitable marker development.
- Limited by availability of compatible fluorescent markers for target systems.
