Quantitative Live Cell Imaging of Nuclear Shape and Chromatin Dynamics During Development and Environmental Stress in Arabidopsis thaliana Root
June 5th, 2026
This process uses confocal microscopy in conjunction with a fluorescently labeled nucleus and chromatin marker line in Arabidopsis thaliana to simultaneously record nucleus and chromatin dynamics in Arabidopsis roots and investigate changes in these dynamics in response to developmental and environmental cues.
Our research investigates genes and mechanisms that enable long-term root recovery from osmotic stress to fortify crops against drought. Although developed for arabidopsis roots, this protocol can be adapted to systems with fluorescent protein marker lines. Obtain the growth plates containing Arabidopsis thaliana seedlings prepared for imaging.
Pipette 60 microliters of pure water onto a glass slide. Lay three six-day-old A.thaliana seedling roots into the water. Place a 22x22 millimeter number 1.5 cover slip over the roots without covering the leaves.
In confocal microscope settings, set the laser exposure time to 1, 000 milliseconds. In the laser combiner settings, set the 488 nanometer laser to 50%for green fluorescent protein, or GFP, imaging. Set the 561 nanometer laser to 50%for red fluorescent protein, or RFP, imaging.
Image the sample using a 20X objective lens without optical zoom. Set the Z slice step size to 0.3 micrometers. Set the imaging interval to one minute, and the total imaging duration to 10 minutes.
Locate the roots and select up to 20 Z slices. Capture 16-bit images. Start the time lapse acquisition and save the time lapse as a TIFF file using lossless compression.
Move the acquired data to a computer for analysis. Open the ImageJ or Fiji software. Click and drag the VSI file onto the main ImageJ or Fiji application window.
In the Bio Formats Import Options prompt, click OK in the bottom right. Then in the Bio Format Series Options prompt, ensure Series 1 is selected. Click OK in the bottom left.
Once the image is loaded, split the signals by selecting Image. Select Color and choose Split Channels. View the separate windows generated for GFP-labeled nuclei and RFP-labeled chromatin.
Determine which of the two image windows corresponds to chromatin. After clicking the chromatin image window, apply the Z project operation by selecting Image, choosing Stacks, and then clicking Z Project from the main ImageJ window. Change the Projection Type to Max Intensity.
Ensure that All Time Frames is active, and click OK to generate the projection. Open the contrast menu by pressing Control Shift C, or by selecting Image and choosing Adjust, and then clicking Brightness or Contrast from the main ImageJ window. Slide the Minimum bar to the left until the chromatin is clearly visible.
Navigate to the plugins. Click the down arrow in the Plugins menu to scroll to the Tracking option. Select Tracking and click TrackMate.
Confirm that the calibration settings are accurate and click Next. Select Thresholding Detector and click Next. Move to the last frame of the time lapse on the image window.
Click Auto next to the Intensity Threshold and click Preview. Adjust the Intensity Threshold and click Preview. Repeat the adjustment and preview process until all chromatin is included with minimal noise, and click Next.
Allow the initial thresholding detection to complete, and click Next. Manually adjust the threshold until only chromatin is visible, and click Next. Adjust the fluorescence image display and click Next.
Select Simple LAP Tracker and click Next. Set the Linking Max Distance to 5 microns, Gap Closing Max Distance to 15 microns, Gap Closing Maximum Frame Gap to 1, and click next. Allow tracking to complete.
Observe the tracks displayed in the original image window and click Next. Press the green plus button in the bottom left corner to create a filter. Select number of spots in track mode.
Adjust the threshold slider. Ensure the filter mode is set to Above, and then click Next. Click the Tracks button at the bottom of the Display Options page.
Select Export to CSV. Ignore the built-in plotting page and click Next. Review the TrackMate analysis results.
Click Capture Overlay and then choose Execute. Save the video by selecting File and clicking Save As, and then choosing GIF. Salt-treated root cells appeared stressed, and centromeric foci appeared larger than in control conditions.
Chromatin speed was significantly higher in control conditions than in 100-millimolar sodium chloride-treated conditions. This protocol enables tracking and quantification of fluorescently-labeled protein movement over time in arabidopsis roots. The most important challenge in this protocol is obtaining high-quality images, as photo bleaching and sample drift can make tracking difficult.
Future studies should investigate the mechanisms underlying the reduced chromatin movement observed in our study.
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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.
