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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.

Two-Dimensional Microscopy in Microbiology

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Two-dimensional (2D) microscopy encompasses a range of optical techniques that capture images within a single focal plane, offering detailed representations of microscopic structures. These techniques are essential in biological and medical research, enabling the visualization of cellular and subcellular structures with different levels of contrast and specificity.There are several major types of 2D microscopy, each with strengths and applications.Bright-Field MicroscopyBright-field microscopy...

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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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Related Experiment Video

Updated: Jun 20, 2026

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Published on: March 24, 2014

Two-photon excitation STED microscopy.

Gael Moneron¹, Stefan W Hell

¹Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

|August 19, 2009

Summary

This summary is machine-generated.

We achieved sub-diffraction resolution using combined two-photon excitation (TPE) and stimulated-emission depletion (STED) fluorescence microscopy. This novel method enhances imaging resolution for nanoparticles and cellular structures beyond the diffraction limit.

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Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Published on: July 6, 2019

Area of Science:

Biophysics
Optical Microscopy
Cell Biology

Background:

Traditional fluorescence microscopy is limited by the diffraction barrier, restricting resolution.
Two-photon excitation (TPE) microscopy offers advantages in deep tissue imaging but still faces diffraction limitations.
Stimulated-emission depletion (STED) microscopy is a super-resolution technique that overcomes the diffraction limit.

Purpose of the Study:

To merge TPE microscopy with STED for enhanced resolution.
To demonstrate a practical laser setup for combined TPE-STED.
To validate the improved resolution using biological samples.

Main Methods:

Implementation of a combined laser system using a short-pulse laser for TPE and a continuous-wave (CW) laser for STED.
Imaging of fluorescent nanoparticles to assess resolution limits.
Imaging of immunostained NF kappaB in mammalian cell nuclei.

Main Results:

Achieved sub-diffraction resolution below 50 nm for fluorescent nanoparticles.
Obtained approximately 70 nm resolution for NF kappaB in cell nuclei.
Demonstrated a 4-5.4-fold improvement in resolution compared to the diffraction limit.

Conclusions:

The combined TPE-STED approach provides a powerful method for super-resolution imaging.
This technique offers an easy-to-implement solution for enhanced microscopy resolution.
The demonstrated resolution improvements are significant for visualizing subcellular structures and molecular interactions.