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Super-resolution Fluorescence Microscopy

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...

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Updated: Nov 25, 2025

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Published on: October 31, 2020

Implementation of a 4Pi-SMS super-resolution microscope.

Jingyu Wang¹, Edward S Allgeyer², George Sirinakis²

¹Department of Engineering Science, University of Oxford, Oxford, UK.

Nature Protocols

|December 17, 2020

Summary

This summary is machine-generated.

This protocol details establishing 4Pi-single-molecule switching (SMS) super-resolution microscopy. It guides researchers in building and operating the instrument for high-resolution 3D cell imaging.

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Super-Resolution Live Cell Imaging of Subcellular Structures

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Last Updated: Nov 25, 2025

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

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Published on: October 31, 2020

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Super-Resolution Live Cell Imaging of Subcellular Structures

Super-Resolution Live Cell Imaging of Subcellular Structures

Published on: January 13, 2021

Area of Science:

Biophysics
Optical Microscopy
Cell Biology

Background:

Single-molecule switching (SMS) fluorescence microscopy, also known as single-molecule localization microscopy, has revolutionized cell biology imaging.
The 4Pi geometry, utilizing opposing objectives, enhances numerical aperture, enabling higher resolution imaging.

Purpose of the Study:

To provide a comprehensive protocol for establishing 4Pi-SMS super-resolution microscopy in research laboratories.
To enable researchers to achieve nanoscale 3D resolution imaging of cellular structures.

Main Methods:

Detailed instructions for assembling and aligning the optomechanical components of a 4Pi-SMS instrument.
Guidance on sample preparation (fluorescent beads), instrument operation, and image data analysis for 3D reconstruction.
Includes a troubleshooting guide and examples of expected results.

Main Results:

Demonstrates 3D resolution down to 10 nm over entire cellular volumes using 4Pi geometry and interferometric detection.
Provides a practical framework for researchers to implement advanced super-resolution microscopy techniques.
Successful acquisition of high-quality biological images after approximately 12 months of instrument construction.

Conclusions:

The protocol facilitates the adoption of 4Pi-SMS microscopy, advancing the field of super-resolution imaging.
Enables detailed visualization of cellular structures at the nanoscale, crucial for understanding biological processes.
Empowers researchers to perform cutting-edge 3D super-resolution microscopy in their own labs.