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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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Related Experiment Video

Updated: Jun 28, 2025

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Published on: July 21, 2017

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ

Haohan Song¹, Chaoqing Dong¹, Jicun Ren¹

¹School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China.

Analytical Chemistry

|April 22, 2024

Summary

This study introduces fluorescence cross-correlation spectroscopy (FCCS) to simultaneously monitor multiple autophagy processes and assess autophagic flux in single cells. This new method efficiently distinguishes autophagic structures and regulators, overcoming limitations of traditional imaging techniques.

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Last Updated: Jun 28, 2025

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

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Published on: July 21, 2017

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A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

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Published on: July 18, 2011

Area of Science:

Cell Biology
Biophysics

Background:

Autophagy is a crucial cellular process for maintaining homeostasis, but its dysregulation is linked to diseases like cancer.
Current fluorescence imaging methods struggle to simultaneously monitor multiple stages of autophagy and assess autophagic flux.
Developing advanced techniques is essential for a deeper understanding of autophagy dynamics.

Purpose of the Study:

To develop and validate a novel method for simultaneously monitoring multiple autophagic processes.
To assess autophagic flux in single cells using fluorescence cross-correlation spectroscopy (FCCS).
To overcome the limitations of conventional fluorescence imaging in studying autophagy.

Main Methods:

Utilized *in situ* fluorescence cross-correlation spectroscopy (FCCS) for real-time monitoring.
Engineered microtubule-associated protein 1A/1B-light chain 3B (LC3B) fused with mCherry and enhanced green fluorescent protein (EGFP).
Introduced a new parameter, "delivery efficiency of autophagosome (DE_AP)," to quantify autophagic flux based on cross-correlation (CC) values.

Main Results:

FCCS successfully distinguished three distinct autophagic structures based on diffusion time (τ_D).
The method accurately assessed induced autophagic flux and differentiated between various autophagy regulators.
FCCS demonstrated superior resolution compared to traditional imaging, unaffected by Brownian motion or cytoplasmic fluorescent monomers.

Conclusions:

FCCS provides an efficient and high-resolution platform for simultaneously monitoring multiple autophagic processes and autophagic flux.
This technique offers significant advantages over conventional methods for studying autophagy dynamics in single cells.
The developed method holds promise for advancing research in autophagy-related diseases and drug discovery.