拟南芥核单分子定位显微镜的简化方案
May 8th, 2026
该方案为单分子定位显微镜成像提供了高效的工作流程，利用优化的分离、固定和标记，实现染色质和RNA聚合酶II的可靠纳米尺度可视化。该方法可轻松应用于其他染色质相关蛋白或组蛋白修饰，实现高分辨率成像。
我的研究重点是表观基因组编辑以及利用超分辨率显微镜可视化拟南南菊核中的染色质组织。现有方法尚未适用于STORM成像。该方案提升了核的纯度、标记一致性、样品装载和成像稳定性。
它适用于拟南芥及其他植物系统，用于高分辨率研究核组织、染色质结构和表观遗传学。首先，将一个六孔培养板放在排气罩下方的冰上。将固定液中体积占4%、甲醇稳定甲醛0.1%、Triton X100和PBS混合。
在每个排气罩下的井中加入大约四毫升冰冷固定液。用干净的产钳取出发芽后8至15天之间的拟南芥幼苗，取出半浓度的Murashige和Skoog实心培养基。立即将幼苗浸入固定溶液中，每孔放置约20株幼苗。
每个样品使用三口井以获得足够的材料。将盘子放入装满冰块的真空腔室并关闭。在负0.6巴到零下0.8巴之间进行真空循环，持续15分钟，以去除滞留空气，让固定剂深入植物组织。
然后，慢慢释放真空。混合样品后再用真空加热15分钟。在排气罩下方，用移液管取出固定液。
用5毫升冰冷PBS轻轻搅拌，每口井洗三次，每次10分钟。洗涤后，取出PBS。用镊子将固定组织转移到120×120毫米的培养皿中，并向固定幼苗添加50微升核分离缓冲液。
用刀片切开固定的组织。用同一把刀片，将切碎的纸巾刮到培养皿的一边。加入600微升核分离缓冲液，并冲洗粘附在刀刃上的组织。
将组织浆液转移到2毫升的Dounce均质管中，用较小的杵冲刺5到10次。然后用较大的杵在管内平滑移动，通常需要5到10次。将均质样品通过30微米网状滤网过滤到50毫升的管子中。
将过滤液转移到一根新的1.5毫升管中。在800G下用最低斜坡离心10分钟，用于加速和减速。离心后，可以看到绿色组织碎片中可见一小颗白色颗粒。
丢弃上清液，将沉淀重新悬浮在600微升的核分离缓冲液中。再次离心10分钟，800克，去除上清液。在完全洗净绿色杂质后，将分离的核悬浮液以四摄氏度储存，最多一周，并用所需的抗体对核进行免疫染色。
将体积比1.5%的低熔点琼脂糖溶液以90摄氏度加热一分钟，使粉末溶解并获得透明溶液。短暂旋转以确保完全混合。冷却溶液并保持在37摄氏度后再进行，以避免葡萄糖氧化酶缓冲液被热变性。
当低熔点琼脂糖溶液达到37摄氏度后，向低熔点琼脂糖中加入100毫莫拉咰基乙胺和葡萄糖氧化酶缓冲液，达到最终浓度的1倍，并轻柔混合。将10微升核混合物沉积在经过臭氧处理的1.5H覆盖层上，该涂层在紫外臭氧清洗系统中处理了20分钟。等待三分钟，让原子核沉积在覆盖层上。
然后，去除多余的液体，等待两分钟。在盖片上加入10微升低熔度琼脂糖，立即用显微镜载玻片覆盖。用硅胶密封盖子。
在一个小培养皿中用微量移液器头混合等量的溶液A和溶液B。将混合物涂抹在盖片边缘以密封样品。让硅胶密封剂硬化五分钟。
把样品载玻片装在显微镜上，找到焦点。定位并聚焦一个原子核。如果有，激活维持焦点模块以确保长时间采集时的稳定性。
选择一个区域，将一颗或几颗纳米金刚石放置在原子核附近，以改善重建过程中的漂移修正。对于双色成像，先从较长波长的通道采集，然后切换到较短波长的通道以完成成像。在ThunderSTORM中使用加权最小二乘估计的高斯拟合处理原始图像堆栈，生成重建图像。
在30纳米以下应用基于σ和不确定度的滤波，以保持准确定位并去除噪声。最后，使用ThunderSTORM交叉相关或COMET算法进行样本漂移校正，确认重建图像中的最小漂移。从15天龄野生型幼苗中分离出核，免疫标记AF647结合二级抗体，并用Hoechst H33258对抗染色。
共焦点成像显示多个分离且保存良好的核，碎片极少，色心和核仁可清晰区分。RNA聚合酶II信号在色胞和核仁中缺失。绕射极限最大强度投影及相应SMLM重建揭示了RNA聚合酶II和DNA标记的结构细节，包括放大的兴趣区域显示出更细微的结构。
基于σ和不确定性的定位过滤改进了RNA聚合酶II和DNA信号的定义，相较于检测到的定位。漂移修正后的进一步改进也维持了这一改进的结构。基于西格玛值的滤波去除了拟合宽度过小或过大的位置化。
傅里叶环相关分析显示，AF647通道的分辨率在滤波后从90.1纳米提升到47.7纳米，而JF549通道的分辨率从56.6纳米提升到27.9纳米。在减少碎片和背景荧光的同时保持核完整性，对于实现高质量单分子成像至关重要。未来的研究有望将核结构与环境或发育条件下的基因调控联系起来。
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Overview
This article introduces a streamlined and reproducible protocol for Single-Molecule Localization Microscopy (SMLM), specifically direct stochastic optical reconstruction microscopy (dSTORM), to visualize chromatin architecture in nuclei isolated from Arabidopsis thaliana. The workflow addresses technical challenges in plant sample preparation, enabling nanoscale imaging of chromatin domains, histone modifications, and nuclear organization with enhanced clarity and reproducibility.
Key Study Components
Area of Science
- Plant cell biology
- Super-resolution microscopy
- Chromatin organization
Background
- Confocal fluorescence microscopy is limited by diffraction, restricting resolution in chromatin studies.
- Super-resolution techniques like SMLM overcome these limitations, providing nanoscale insights.
- dSTORM is widely used in mammalian systems but less so in plants due to sample preparation challenges.
- Improved protocols are needed to facilitate SMLM in plant nuclei.
Purpose of Study
- To develop a reproducible workflow for SMLM imaging of plant nuclei.
- To enable high-resolution visualization of chromatin modifications and nuclear architecture in Arabidopsis thaliana.
- To lower technical barriers for applying super-resolution microscopy in plant biology.
Methods Used
- Fixation of Arabidopsis seedlings to preserve nuclear morphology.
- Gentle tissue chopping and centrifugation to enrich for intact nuclei.
- Fluorophore labeling of isolated nuclei in liquid medium.
- Immobilization of nuclei on low-melting agarose pads for imaging stability.
Main Results
- The protocol minimizes background fluorescence and improves labeling consistency.
- Enhanced reproducibility is achieved across biological replicates.
- Preparations allow clear visualization of chromatin modifications and nuclear architecture at the nanoscale.
- The workflow facilitates studies of epigenetic regulation and nuclear topology in plants.
Conclusions
- This protocol establishes a methodological foundation for SMLM imaging in plant systems.
- It bridges the gap between plant and mammalian cell biology in super-resolution imaging.
- The approach opens new opportunities to study genome regulation and nuclear architecture in response to developmental and environmental cues in plants.
Frequently Asked Questions
Executive Industry Relevance
Super-resolution imaging of chromatin architecture in plant nuclei addresses a critical gap in understanding epigenetic regulation and nuclear organization at the nanoscale. This streamlined SMLM protocol for Arabidopsis nuclei enables reproducible, high-resolution visualization, supporting predictive confidence in chromatin state analysis. The approach enhances early discovery and mechanistic de-risking for plant-based R&D portfolios.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables nanoscale interrogation of chromatin domains and histone modifications in plant nuclei.
- Supports functional validation of epigenetic targets by visualizing nuclear architecture changes.
- Facilitates mechanistic de-risking through direct observation of chromatin organization.
- Improves predictive confidence in linking nuclear structure to gene regulation.
Screening & Assay Development
- Provides standardized sample preparation for reproducible super-resolution imaging workflows.
- Delivers quantitative, high-clarity readouts of chromatin modifications for assay development.
- Enables consistent labeling and imaging across biological replicates, supporting screening scalability.
- Prepares validated plant nuclear systems for downstream compound or genetic screening.
Translational & Preclinical Research
- Aligns chromatin imaging outputs with translational biomarker discovery in plant systems.
- Supports continuity from discovery through preclinical validation of epigenetic interventions.
- Enables risk-adjusted advancement by clarifying nuclear topological changes under different conditions.
- Provides mechanistic insights relevant to environmental and developmental response studies.
Pipeline & Workflow Integration
This protocol integrates into the discovery continuum from early chromatin hypothesis testing to preclinical validation of epigenetic mechanisms in plants.
- Discovery Biology: Supports hypothesis-driven analysis of chromatin organization and nuclear architecture.
- Screening: Delivers reproducible, quantitative imaging outputs for assay standardization.
- Analytics: Enables precise measurement of chromatin modifications and nuclear features for comparative studies.
- Translational Research: Bridges discovery and preclinical research by enabling biomarker alignment in plant systems.
- Enterprise Reuse: Establishes a reusable imaging workflow adaptable to diverse plant research applications.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces ambiguity in chromatin state analysis.
- Operational Value: Standardizes sample preparation and imaging, enhancing reproducibility and scalability.
- Strategic Value: Improves go/no-go decisions for epigenetic target programs in plant R&D.
- Portfolio Impact: Enables risk-adjusted prioritization of plant-based discovery and translational projects.
Implementation Considerations
- Requires expertise in super-resolution microscopy and plant nuclear isolation.
- Needs access to dSTORM-capable imaging platforms and fluorophore labeling reagents.
- Demands cross-team standardization of fixation, labeling, and immobilization protocols.
- May require adaptation for different plant species or tissue types.
- Sample stability and background minimization are critical for reproducible outputs.
