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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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Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

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Real-Time and Site-Specific Perturbation of Dynamic Subcellular Compartments Using Femtosecond Pulses.

Seohee Ma^1,2, Bin Dong^1,2, Matthew G Clark¹

¹James Tarpo Jr. and Margaret Tarpo Department of Chemistry Purdue University 560 Oval Dr. West Lafayette 47907 IN USA.

|July 16, 2025

Summary

This study introduces femtosecond real-time precision opto-control (fs-RPOC), a new method for precise subcellular laser manipulation. Fs-RPOC enables automated targeting and real-time perturbation of dynamic biomolecules within cells.

Keywords:

femtosecond laser low‐density plasma microsurgery mitochondria pulse‐picking reactive oxygen species real‐time precision opto‐control

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Published on: August 22, 2017

Area of Science:

Cell Biology
Biophotonics
Laser Physics

Background:

Femtosecond (fs) lasers offer high precision for subcellular applications via nonlinear multiphoton absorption.
Current fs laser delivery lacks automated targeting for dynamic molecules and simultaneous imaging/perturbation.
Existing methods hinder real-time analysis of cellular responses to laser-induced perturbations.

Purpose of the Study:

To develop a system for automated, chemically selective, and real-time subcellular perturbation using fs lasers.
To enable precise control over laser parameters for targeted molecular modulation within specific organelles.
To investigate cellular responses to fs laser interactions at the subcellular level.

Main Methods:

Integration of a laser scanning microscope with a closed-loop feedback system for fs-RPOC.
Utilizing a pulse-picking method for independent control of fs laser average and peak power.
Targeting mitochondria to study localized molecular responses to laser-induced events.

Main Results:

Fs-RPOC demonstrated superior spatial precision and fast response times for subcellular microsurgery.
Automated, selective perturbation of single- and sub-organelle targets was achieved.
Site-specific molecular responses, including reactive oxygen species formation and H2O2 diffusion, were observed in mitochondria.

Conclusions:

Fs-RPOC provides unprecedented control for precise molecular and organelle regulation.
The system enables new insights into fs laser interactions with subcellular compartments.
This technology advances optical microscopy, phototherapy, and optogenetics through real-time cellular control.