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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy
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Variable-Angle Epifluorescence Microscopy for Single-Particle Tracking in the Plant ER.

Charlotte Pain1, Christopher Tynan2, Stanley W Botchway2

  • 1Endomembrane Structure and Function Research Group, Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK.

Methods in Molecular Biology (Clifton, N.J.)
|February 27, 2024
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Summary

This study presents methods for single-particle tracking in plant endoplasmic reticulum using variable-angle epifluorescence microscopy. These techniques enable dynamic observation of biomolecule movement within challenging plant cells.

Keywords:
Automated single-particle trackingEndoplasmic reticulumSingle-particle trackingTrackMateVAEMmEOSpaGFP

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Last Updated: Jul 2, 2025

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Area of Science:

  • Plant cell biology
  • Biophysics
  • Microscopy techniques

Background:

  • Observing single biomolecules in plant endoplasmic reticulum (ER) is crucial for understanding protein complex formation and molecular transport.
  • Plant cells present significant challenges for live-cell imaging due to their size, structure, and autofluorescence.

Purpose of the Study:

  • To develop and describe methods for single-particle tracking (SPT) of biomolecules within the plant ER.
  • To overcome the challenges of imaging in plant cells using advanced microscopy techniques.

Main Methods:

  • Utilized variable-angle or highly inclined epifluorescence microscopy (VAEM) for enhanced imaging.
  • Employed transient expression in tobacco cells for biomolecule observation.
  • Developed methods for preparing plant samples for VAEM and analyzing single-particle tracks.

Main Results:

  • Demonstrated the feasibility of observing single-particle dynamics in the plant ER using VAEM.
  • Showcased the use of tuneable fluorophores for dynamic control of fluorophore density.
  • Established robust methods for determining and analyzing single-particle tracks from time-series VAEM data.

Conclusions:

  • The described methods facilitate successful SPT in the plant ER, overcoming inherent cellular challenges.
  • These techniques provide new avenues for studying protein-protein interactions, metabolons, and molecular transport in plants.
  • The study offers practical protocols for researchers interested in plant cell dynamics.