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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: Aug 26, 2025

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Published on: April 28, 2014

Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.

Joshua J Rennick¹, Cameron J Nowell¹, Colin W Pouton¹

¹Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Nature Communications

|October 12, 2022

Summary

This summary is machine-generated.

This study introduces pH dependant fluorescent lifetime imaging microscopy (pHLIM) combined with deep learning for precise sub-cellular pH measurement. This novel method accurately quantifies pH in vesicles, aiding drug action and disease research.

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Last Updated: Aug 26, 2025

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Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

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Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

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Published on: February 21, 2019

Area of Science:

Cell biology
Biophysics
Pharmacology

Background:

Sub-cellular pH is crucial for cellular processes like metabolism and drug delivery.
Existing pH measurement methods using fluorophores are often uncertain and require multiple probes.

Purpose of the Study:

To develop a precise and quantitative method for measuring sub-cellular pH.
To overcome limitations of current intensity-based pH measurements.

Main Methods:

Combined pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning.
Engineered the pH-sensitive protein mApple for localization in cytosol, endosomes, and lysosomes.
Utilized pHLIM to detect drug-induced pH changes (bafilomycin A1, chloroquine).

Main Results:

Accurate quantification of sub-cellular pH in individual vesicles.
Demonstrated rapid detection of pH changes induced by specific drugs.
Showed polyethylenimine does not cause a proton sponge effect in endocytic vesicles.

Conclusions:

pHLIM offers a simple, quantitative approach for sub-cellular pH measurement.
This method can enhance understanding of drug mechanisms and disease progression.
pHLIM provides a valuable tool for cell biology and drug development research.