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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 developed.

Confocal Fluorescence Microscopy

Confocal Fluorescence Microscopy

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

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Updated: Jun 24, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging.

Sebastian van de Linde¹, Ulrike Endesfelder, Anindita Mukherjee

¹Bielefeld Institute for Biophysics and Nanoscience, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.

Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology

|April 2, 2009

Summary

This summary is machine-generated.

This study presents a novel multicolor fluorescence imaging technique using photoswitching of common organic fluorophores. This method achieves subdiffraction-resolution imaging for biological samples, offering approximately 20 nm optical resolution.

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Area of Science:

Biophysics
Optical Microscopy
Molecular Imaging

Background:

Standard fluorescence microscopy is limited by diffraction, hindering visualization of fine cellular structures.
Achieving multicolor imaging at the nanoscale requires advanced techniques beyond conventional optical resolution.

Purpose of the Study:

To develop a general approach for multicolor subdiffraction-resolution fluorescence imaging.
To utilize photoswitching of standard organic fluorophores for enhanced imaging capabilities.

Main Methods:

Employing photoswitchable organic fluorophores (e.g., ATTO520, ATTO565) in aqueous buffers.
Exploiting reversible transitions between fluorescent and nonfluorescent states via triplet radical anions and molecular oxygen.
Controlling fluorophore residency time in the fluorescent state using excitation intensity and reducing agent concentration.

Main Results:

Demonstrated multicolor photoswitching microscopy with subdiffraction-resolution.
Achieved approximately 20 nm optical resolution for detailed imaging.
Successfully imaged cytoskeletal networks and quantified mitochondrial membrane proteins.

Conclusions:

The developed photoswitching approach enables multicolor super-resolution imaging with standard fluorophores.
This technique offers a versatile tool for high-resolution visualization and molecular quantification in biological systems.