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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...

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,...

Total Internal Reflection Fluorescence Microscopy

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.

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Related Experiment Video

Updated: Jun 17, 2026

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

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Published on: October 9, 2014

Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging.

Qifeng Li¹, Sherry S H Wu, Keng C Chou

¹Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada.

Biophysical Journal

|December 17, 2009

Summary

This summary is machine-generated.

We developed a super-resolution microscope combining two-photon excitation fluorescence (2PEF) and stimulated emission depletion (STED) microscopy. This advanced imaging technique achieves subdiffraction-limit resolution for enhanced cell visualization.

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Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Two-photon excitation fluorescence (2PEF) microscopy offers intrinsic 3D resolution and reduced photobleaching.
Achieving subdiffraction-limit resolution is crucial for visualizing fine cellular structures.

Purpose of the Study:

To demonstrate subdiffraction-limit resolution for green-fluorescent-protein-tagged cell imaging.
To integrate 2PEF and stimulated emission depletion (STED) microscopy for enhanced imaging capabilities.

Main Methods:

Development of a hybrid microscope combining 2PEF and STED microscopy.
Imaging of green-fluorescent-protein-tagged caveolar vesicles in cells.

Main Results:

The integrated microscope achieved subdiffraction-limit resolution, surpassing the diffraction limit of conventional 2PEF microscopy (approx. 250 nm).
The effective point-spread function (PSF) full width at half-maximum (FWHM) was measured to be approximately 54 nm.
Successfully resolved cellular structures closer than the diffraction limit.

Conclusions:

The combined 2PEF and STED microscopy provides superior resolution for detailed cell imaging.
This technique enables visualization of nanoscale cellular components with unprecedented clarity.