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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,...

Total Internal Reflection Fluorescence Microscopy

Total Internal Reflection Fluorescence Microscopy

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: Jul 2, 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

Intensity calibration and shading correction for fluorescence microscopes.

Michael A Model¹

¹Kent State University, Kent, Ohio, USA.

Current Protocols in Cytometry

|September 5, 2008

Summary

This summary is machine-generated.

This study introduces a simple, inexpensive method for standardizing fluorescence microscopy using concentrated dye solutions. This approach ensures reproducible results for intensity calibration and shading correction in image cytometry.

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Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

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Published on: December 1, 2016

Area of Science:

Biophysics
Microscopy techniques
Analytical chemistry

Background:

Standardization is crucial for accurate image cytometry.
Current methods for intensity calibration and shading correction can be complex.
Need for reproducible and cost-effective standardization techniques.

Purpose of the Study:

To present a straightforward method for standardizing fluorescence microscopy.
To enable reliable intensity calibration and shading correction.
To offer a reproducible and inexpensive solution for image cytometry.

Main Methods:

Utilizing a drop of highly concentrated fluorophore solution between slide and coverslip.
Creating a spatially uniform fluorescent sample.
Applying the technique to both wide-field and confocal scanning fluorescence microscopy.

Main Results:

Achieved a reproducible quantum yield.
Demonstrated resistance to photobleaching.
Developed an inexpensive and straightforward standardization technique.

Conclusions:

The presented method offers a practical and effective approach to fluorescence microscopy standardization.
This technique enhances reproducibility and accuracy in image cytometry.
Suitable for various fluorescence microscopy setups, including wide-field and confocal.