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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...

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Proteins targeted to the nucleus carry short stretches of amino acid sequences called the nuclear localization signal or NLS. Classical nuclear localization signals are of two types: monopartite and bipartite NLS. Monopartite classical NLS (cNLS) consists of a single cluster of 4-8 amino acids. Bipartite cNLS consists of two clusters of 2-3 amino acids and a 9-12 residue long proline-rich linker bridging the two clusters. Signal clusters are rich in positively charged amino acids such as...

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Related Experiment Video

Updated: Jan 18, 2026

Single-Molecule Imaging of Nuclear Transport

Single-Molecule Imaging of Nuclear Transport

Published on: June 9, 2010

Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

Jervis Vermal Thevathasan^1,2, Maurice Kahnwald¹, Konstanty Cieśliński¹

¹EMBL, Cell Biology and Biophysics, Heidelberg, Germany.

|September 29, 2019

Summary

This summary is machine-generated.

New cell lines use nuclear pore complexes as standards to improve microscopy quality control. This enables precise calibration, labeling efficiency quantification, and molecular counting for better imaging results.

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Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Published on: September 13, 2022

Area of Science:

Cell Biology
Microscopy
Biophysics

Background:

Quantitative fluorescence and superresolution microscopy face limitations due to data quality issues and artifacts.
Biologically relevant control samples are crucial for benchmarking and optimizing microscopy techniques, labels, and imaging parameters.

Purpose of the Study:

To develop and validate novel in situ reference structures for characterizing microscopy performance.
To enable accurate calibration, quality control, and quantitative measurements in advanced imaging modalities.

Main Methods:

Creation of four genome-edited cell lines with endogenous labeling of nucleoporin Nup96 using mEGFP, SNAP-tag, HaloTag, and mMaple.
Utilizing the stereotypic arrangement of proteins within the nuclear pore complex as reference structures.
Application of these cell lines across various microscopy modalities.

Main Results:

Demonstrated the utility of the engineered cell lines as three-dimensional resolution standards for calibration and quality control.
Enabled the quantification of absolute labeling efficiencies for different tags.
Established precise reference standards for molecular counting in microscopy.

Conclusions:

The developed cell lines provide a robust platform for assessing microscope and label quality.
Facilitates quantitative and absolute measurements, advancing the field of superresolution and fluorescence microscopy.
Empowers researchers to improve the reliability and accuracy of their imaging data.