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Super-resolution Fluorescence Microscopy

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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: Sep 10, 2025

Quantitative Immunofluorescence to Measure Global Localized Translation

Quantitative Immunofluorescence to Measure Global Localized Translation

Published on: August 22, 2017

Non-Uniform Illumination Correction for Quantitative Fluorescence Imaging.

Yue Wang^1,2, Yongqiang Liu^1,2, Beini Sun^1,2

¹Key Laboratory of Laser Life Science, Ministry of Education, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou, Guangdong, China.

Microscopy Research and Technique

|August 27, 2025

Summary

This summary is machine-generated.

Systematic Uniform Fluorescence Image Correction (SUFIC) improves quantitative microscopy by correcting non-uniform illumination. This efficient prospective method requires minimal reference images, enhancing accuracy in fluorescence resonance energy transfer (FRET) imaging.

Keywords:

fluorescence imaging illumination correction illumination uniformity quantitative FRET

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Last Updated: Sep 10, 2025

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Published on: December 9, 2013

Area of Science:

Microscopy and Imaging Technologies
Biophysics
Quantitative Fluorescence Imaging

Background:

Illumination uniformity is critical for accurate quantitative analysis in fluorescence microscopy.
Existing retrospective correction methods struggle with limited image data, while prospective methods require frequent recalibration.
Non-uniform illumination significantly impacts quantitative fluorescence resonance energy transfer (FRET) measurements.

Purpose of the Study:

To theoretically and experimentally analyze the effect of non-uniform illumination on quantitative FRET.
To introduce Systematic Uniform Fluorescence Image Correction (SUFIC), a novel prospective illumination correction method.
To evaluate the efficacy of SUFIC compared to existing methods.

Main Methods:

Theoretical analysis of illumination non-uniformity's impact on FRET.
Development and application of the Systematic Uniform Fluorescence Image Correction (SUFIC) method.
Experimental validation using fluorescent microspheres, Argo-HM slides, and live cells expressing FRET constructs.

Main Results:

SUFIC significantly improved field uniformity in Argo-HM slide images (27.45% to 65.30%).
SUFIC enhanced signal-to-background ratios in FRET images by 12.3%, 7.9%, and 20.9%.
SUFIC requires a minimum of three reference images, substantially fewer than CIDRE and BaSiC methods.

Conclusions:

SUFIC is a highly efficient prospective method for illumination correction in quantitative microscopy.
The method offers improved accuracy and reduced recalibration needs for fluorescence imaging.
SUFIC demonstrates broad applicability across various biological samples and FRET studies.