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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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A noninvasive fluorescence imaging-based platform measures 3D anisotropic extracellular diffusion.

Peng Chen¹, Xun Chen¹, R Glenn Hepfer^1,2

¹Clemson-MUSC Joint Bioengineering Program, Department of Bioengineering, Clemson University, Clemson, SC, USA.

Nature Communications

|March 27, 2021

Summary

This summary is machine-generated.

A new noninvasive imaging technique, light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP), accurately measures 3D molecular diffusion in tissues. This tool overcomes limitations of previous methods and reveals how tissue changes impact diffusion.

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Area of Science:

Biophysics
Biomolecular Engineering
Tissue Engineering

Background:

Diffusion is a fundamental molecular transport process in biology, crucial for understanding tissue function.
Quantifying anisotropic (direction-dependent) diffusion is essential for relating 3D tissue structure to its biochemical environment.
Existing methods for measuring extracellular diffusion are often invasive and provide limited 3D information, leading to controversial findings.

Purpose of the Study:

To develop and validate a noninvasive tool for measuring 3D anisotropic extracellular diffusion of biomolecules.
To assess the capabilities of the new technique in physiological diffusivity ranges.
To investigate the impact of tissue structure and composition on diffusion.

Main Methods:

Development of light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP).
Measurement of 3D diffusion tensors for various biomolecules.
Application of LiFT-FRAP to corneal tissue as a model system.
Comparison with existing invasive 2D diffusion measurement techniques.

Main Results:

LiFT-FRAP successfully measures 3D anisotropic diffusion of biomolecules up to 51 µm²/s, covering physiological ranges.
The technique highlights limitations of current invasive 2D methods, resolving previous diffusion measurement controversies.
LiFT-FRAP demonstrates that diseases and scaffold fabrication induce direction-dependent changes in tissue diffusion.

Conclusions:

LiFT-FRAP is a powerful, noninvasive platform technology for studying molecular diffusion in 3D.
The technique provides critical insights into disease mechanisms and tissue engineering applications.
LiFT-FRAP can advance clinical outcomes by accurately characterizing tissue environments.