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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Published on: June 30, 2018
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Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores.
Verena Richter1, Peter Lanzerstorfer2,3, Julian Weghuber2,3
1Institute of Applied Research, Aalen University, 373430 Aalen, Germany.
International Journal of Molecular Sciences
|September 30, 2020
Summary
We developed a simple super-resolution microscopy technique for live cells using Structured Illumination Microscopy (SIM) and total internal reflection fluorescence (TIRF-SIM). This method allows detailed visualization of membrane protein dynamics, like glucose transporter 4 (GLUT4) translocation.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Super-resolution microscopy is crucial for visualizing subcellular structures.
- Live-cell imaging requires high resolution and minimal phototoxicity.
- Understanding membrane protein dynamics is key in cell signaling.
Purpose of the Study:
- To present a simple and robust super-resolution live-cell microscopy setup.
- To enable visualization of membrane-proximal fluorophores with high resolution.
- To demonstrate the applicability for studying protein translocation.
Main Methods:
- Structured Illumination Microscopy (SIM) with a spatial light modulator (SLM).
- Total Internal Reflection Fluorescence-SIM (TIRF-SIM) for enhanced surface sensitivity.
- Exchangeable objective lenses for versatile illumination modes.
- Live-cell imaging of glucose transporter 4 (GLUT4) translocation.
Main Results:
- Achieved lateral resolution of ~100 nm and axial resolution of ~200 nm.
- SIM visualized intracellular GLUT4 localization.
- TIRF-SIM enabled quantitative evaluation of GLUT4 in the plasma membrane.
- Demonstrated GLUT4 translocation upon insulin stimulation.
Conclusions:
- The developed SIM and TIRF-SIM methods offer a powerful, easy-to-implement approach for super-resolution live-cell microscopy.
- This technique provides valuable insights into membrane protein dynamics and cellular responses.
- The methods facilitate quantitative analysis of molecular events at the plasma membrane.
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