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Related Experiment Video

Updated: Apr 27, 2026

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Published on: November 12, 2020

Nanoscale protein diffusion by STED-based pair correlation analysis.

Paolo Bianchini¹, Francesco Cardarelli², Mariagrazia Di Luca³

¹Nanophysics, IIT-Italian Institute of Technology, Genoa, Italy.

|June 27, 2014

Summary

This summary is machine-generated.

We combined super-resolution microscopy with pair correlation analysis to map molecular diffusion below the optical limit. This novel STED-pCF method revealed nuclear envelope proximity effects on protein mobility, invisible with conventional microscopy.

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Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Understanding molecular mobility within cells is crucial for deciphering biological processes.
Conventional microscopy techniques are limited by the optical diffraction limit, hindering detailed analysis of nanoscale diffusion.

Purpose of the Study:

To develop and validate a super-resolution technique combining stimulated emission depletion (STED) microscopy and pair correlation function analysis (pCF).
To achieve spatial resolution below the optical diffraction limit for diffusion mapping in living cells.
To investigate the impact of nuclear envelope proximity on protein diffusion.

Main Methods:

Integration of STED microscopy with pCF analysis (STED-pCF) for enhanced spatial resolution.
Application of STED-pCF to model systems: Capsid Like Particles (CLPs) and monomeric fluorescent proteins in CHO cells.
Comparison of STED-pCF resolution (approx. 110 nm) with conventional confocal microscopy.

Main Results:

Achieved a twofold improvement in spatial resolution compared to confocal microscopy.
Successfully mapped diffusion features of proteins near the nuclear envelope in living cells.
Unveiled previously undetected local diffusion barriers and a slow diffusion component (500-700 nm from nuclear envelope).

Conclusions:

STED-pCF is a powerful tool for super-resolution diffusion mapping in biological systems.
Nuclear envelope proximity significantly influences protein mobility, introducing barriers and distinct diffusion components.
This method provides insights into molecular transport dynamics invisible to conventional optical methods.