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Updated: Mar 23, 2026
07:42
Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection
Published on: February 24, 2026
585
Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching.
Yan Fu1, Peter W Winter2, Raul Rojas3
1Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892;
Summary
This study introduces a multiangle total internal reflection fluorescence (TIRF) microscope for superresolution optical sectioning. The method achieves 20-nm axial resolution, enabling detailed 3D imaging and visualization of cellular processes like endocytosis.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Superresolution microscopy is crucial for visualizing cellular structures.
- Total Internal Reflection Fluorescence (TIRF) microscopy offers high resolution near the coverslip.
- Limitations exist in resolving structures at greater depths due to signal overlap.
Purpose of the Study:
- To develop a superresolution optical sectioning technique using a multiangle TIRF microscope.
- To achieve high axial resolution for 3D imaging of biological samples.
- To investigate the dynamics of clathrin-mediated endocytosis.
Main Methods:
- Utilized a multiangle TIRF microscope, altering excitation light incident angles.
- Sequentially imaged and photobleached fluorescent molecules at different depths.
- Employed Bayesian analysis for lateral superresolution within sections.
Main Results:
- Achieved 20-nm axial resolution in 3D imaging of biological samples.
- Successfully visualized the dynamics of epidermal growth factor (EGF) ligand and clathrin during endocytosis.
- Demonstrated lateral superresolution below 100 nm within individual optical sections.
Conclusions:
- The multiangle TIRF method enables effective superresolution optical sectioning and 3D imaging.
- This technique provides new insights into the spatiotemporal dynamics of endocytosis.
- The developed method enhances the capabilities of fluorescence microscopy for biological research.
